Methods and compositions for treating viral diseases

ABSTRACT

The invention provides a method for treating viral infections and coinfections through the use of inhibitory agents that prevent a unique viral structural protein motifs from binding to host proteins from the clathrin adaptor proteins family and subsequently preventing viral replication.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.14/362,993, filed Jun. 5, 2014, which is a U.S. National filing of andclaims priority to PCT/US2012/068167, filed Dec. 6, 2012, which claimspriority to U.S. Provisional Patent Application No. 61/567,491, filedDec. 6, 2011. Priority to each of the above referenced applications isclaimed and the content of each application is incorporated herein byreference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under contract AI079406awarded by the National Institutes of Health. The Government has certainrights in this invention.

FIELD OF THE INVENTION

The present invention relates to virology. More specifically, theinvention relates to treatment of viral diseases through inhibition ofinteractions of host cellular clathrin adaptor proteins and viral YXXΦor dileucine motifs or their regulation.

BACKGROUND OF THE INVENTION

Human infections with Lentiviridae, such as HIV, and Flaviviridae, suchas HCV, pose significant challenges to global health. Although potentanti-HCV drugs are in clinical development and response rates tointerferon-based regimens have improved with the inclusion of proteaseinhibitors (PI), resistance, drug-drug interactions, and cumulativetoxicity continue to pose challenges. More effective antiviralstrategies are therefore still in need to combat the HCV pandemics. Inaddition, novel antiviral strategies are needed for inclusion in salvageregimens for treating HIV in patients failing highly activeantiretroviral therapy (HAART) due to resistant virus.

SUMMARY OF THE INVENTION

Briefly described, embodiments of this disclosure include compounds,compositions, pharmaceutical compositions, and methods of treating ahost with a viral infection. In an embodiment, susceptible virusesaccording to the present disclosure include viruses comprising at leastone protein comprising a YXXΦ motif or a dileucine motif, referred toherein as “clathrin AP binding viruses,” Flaviviridae, Lentiviridae,HCV, HIV, co-infections, such as HCV/HIV co-infections, Flaviviridaeother than HCV, clathrin AP binding viruses other than Flaviviridae, orclathrin AP binding viruses other than HCV, methods of treatingreplication of such virus in a host, methods of inhibiting the bindingof a viral structural protein comprising the YXXΦ or dileucine motifs tohost μ subunits of clathrin AP1-AP4 complexes, such as AP2M1, AP1M1,AP3M1, or AP4M1, methods of treating viral hepatitis-related liverfibrosis in a host, and the like.

One exemplary method of treating a host with a viral infection fromclathrin AP binding viruses, Flaviviridae, Lentiviridae, HCV, HIV,co-infections, such as HCV/HIV co-infections, Flaviviridae other thanHCV, clathrin AP binding viruses other than Flaviviridae, or clathrin APbinding viruses other than HCV, among others, may include: administeringto the host a therapeutically effective amount of an inhibiting agent toreduce the viral load in the host. In an embodiment, the inhibitingagent is selected from the group consisting of agents that competitivelyinhibit binding between a viral protein comprising the YXXΦ or dileucinemotifs and a host protein selected from the group consisting of the μsubunits of clathrin AP1-AP4; and agents that inhibit host proteinkinase activity of kinases that modulate the activity of host proteinsselected from the group consisting of μ subunits of clathrin AP1-AP4.One exemplary method of treating viral infection of clathrin AP bindingviruses, Flaviviridae, Lentiviridae, HCV, HIV, co-infections, such asHCV/HIV co-infections, Flaviviridae other than HCV, clathrin AP bindingviruses other than Flaviviridae, or clathrin AP binding viruses otherthan HCV, in a host, among others, may include: administering aninhibiting agent to the host having such viral infection or infections.In an embodiment, the inhibiting agent is selected from the groupconsisting of agents that competitively inhibit binding between a viralstructural protein comprising the YXXΦ or dileucine motifs and a hostprotein selected from the group consisting of AP2M1, and agents thatinhibit host protein kinase activity of kinases that modulate theactivity of host proteins selected from the group consisting of AP2M1and other μ subunits of clathrin AP complexes. In some embodiments,these inhibitors inhibit GAK (cyclin G-associated kinase) or AAK1(adaptor-associated kinase 1), which include compounds such aserlotinib, sunitinib, or PKC-412. Thus, inhibitory agents are of twoclasses: (1) competitive inhibitors of binding; and (2) agents thatinhibit host protein kinase activity of kinases that modulate theactivity of host proteins such as AP2M1 and/or other μ subunits ofclathrin AP complexes.

One exemplary method of inhibiting the binding of the YXXΦ or dileucinemotifs to host polypeptides, among others, includes administering aninhibiting agent to the host having a viral infection. In an embodiment,the inhibiting agent is selected from the group consisting of agentsthat competitively inhibit binding between a viral structural proteincomprising the YXXΦ or dileucine motifs and a host protein selected fromAP2M1 or other μ subunits of clathrin AP complexes, and agents thatinhibit protein kinase activity of kinases that modulate the activity ofhost proteins selected from the group consisting of AP2M1 and/or other μsubunits of clathrin AP complexes.

One exemplary pharmaceutical composition for treating a host having aviral infection of clathrin AP binding viruses, Flaviviridae,Lentiviridae, HCV, HIV, co-infections, such as HCV/HIV co-infections,Flaviviridae other than HCV, clathrin AP binding viruses other thanFlaviviridae, or clathrin AP binding viruses other than HCV, amongothers, may include an inhibiting agent. In an embodiment, theinhibiting agent is selected from the group consisting of agents thatcompetitively inhibit binding between a viral structural proteincomprising the YXXΦ or dileucine motifs and a host protein selected fromAP2M1 and/or other μ subunits of clathrin AP complexes, and agents thatinhibit protein kinase activity of kinases that modulate the activity ofhost proteins secreted from the group consisting of AP2M1 and/or other μsubunits of clathrin AP complexes.

One exemplary composition, among others, includes an inhibiting agent.In an embodiment, the inhibiting agent is selected from the groupconsisting of agents that competitively inhibit binding between a viralprotein comprising the YXXΦ or dileucine motif and a host proteinselected from the group consisting of AP2M1 and/or other μ subunits ofclathrin AP complexes, and agents that inhibit protein kinase activityof kinases that modulate the activity of host proteins selected from thegroup consisting of AP2M1 and/or other μ subunits of clathrin APcomplexes.

In one aspect, the present invention provides a method of treatingHCV-HIV co-infection through administration of an effective amount of anagent that inhibits binding of HCV and HIV to host proteins selectedfrom the group consisting of AP2M1 and other μ subunits of clathrin APcomplexes. In one embodiment, the inhibiting agent inhibits AAK1 or GAK.In another embodiment, the agent is selected from the group consistingof erlotinib, sunitinib, and PKC-412. In another embodiment, the agentprevents binding of a Lentiviridae or Flaviviridae structural proteincontaining a YXXΦ or dileucine motif to a host protein selected from thegroup consisting of AP2M1 and/or other μ subunits of clathrin APcomplexes. In still another embodiment, the agent inhibits AAK.

In another aspect, the invention provides a method of inhibitinginfections by at least a first virus other than HCV, another member ofFlaviviridae, or a member of Lentiviridae, or combinations thereof,through administration to a patient in need an effective amount of anagent that inhibits binding of a structural protein from a viruscontaining a YXXΦ or dileucine motif to a host protein selected from thegroup consisting of AP2M1 and/or other μ subunits of clathrin APcomplexes. In one embodiment, the agent inhibits AAK1 or GAK. In anotherembodiment, the agent is selected from the group consisting oferlotinib, sunitinib, and PKC-412.

In another aspect, the present invention provides a method of screeningfor antiviral agents through contacting a susceptible virus as describedherein or viral protein with a candidate agent and a protein selectedfrom the group consisting of AP2M1 and/or other μ subunits of clathrinAP complexes, and determining an effect of the candidate agent on thebinding of virus to the host protein.

In another aspect, the present invention provides a method of screeningfor antiviral agents through contacting a susceptible virus as describedherein with a candidate agent and a cell expressing a protein selectedfrom the group consisting of AP2M1 and other μ subunits of clathrin APcomplexes, and determining the effect of the candidate agent on viralassembly or budding. In one embodiment, the candidate agent inhibits theactivity of AAK1 or GAK.

In another aspect, the present invention provides a method of inhibitingassembly or budding of clathrin AP binding viruses, Flaviviridae,Lentiviridae, HCV, HIV, co-infections, such as HCV/HIV co-infections,Flaviviridae other than HCV, clathrin AP binding viruses other thanFlaviviridae, or clathrin AP binding viruses other than HCV, in a hostcell through contacting the host cell with an agent that preventsbinding of a viral structural protein containing a YXXΦ or dileucinemotif to a host protein selected from the group consisting of AP2M1 andother μ subunits of clathrin AP complexes. In one embodiment, the agentblocks binding of the viral structural protein to the host protein. Inanother embodiment, the agent competes with the viral structural proteinfor binding to the host protein. In another embodiment, the agentcompetes with the host protein for binding to the viral structuralprotein. In still another embodiment, the method involves screening ofAAK1 and GAK inhibitors for non-Flaviviridae viruses.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the invention. Theinvention may be better understood by reference to one or more of thesedrawings in combination with the detailed description of specificembodiments presented herein:

FIG. 1A-F: Shows a viral core with a YXXΦ motif that binds AP2M1. FIG.1(A) Schematics of core. The location of the identified motif isindicated. FIG. 1(B)-(C) Consensus sequences of YXXΦ motifs fromrepresentative human (B) and viral (C) proteins. FIG. 1(D) The consensussequence of all HCV isolates, clones used in this study, and engineeredcore mutants. FIG. 1(E) Fluorescent images from a microfluidic chip andschematics. Left: AP2M1-V5-his was anchored to the device surface viaits interaction with anti-His antibodies and labeled with anti-V5-FITCantibodies. Middle: T7-tagged core or NS3 were incubated with surfacebound AP2M1 and labeled with anti-T7-Cy3 antibodies. Interactions weretrapped mechanically by MITOMI. Cy3 signal representing bound viralprotein is shown following a wash. Right: an overlay of the Cy3 and FITCsignals, representing bound viral prey to human bait ratio. FIG. 1F. Invitro binding curves of core-T7 and NS3-T7 to surface bound AP2M1. Yaxis represents bound viral protein to surface bound AP2M1 ratio.

FIG. 2A-E: Shows a viral core binding AP2M1 in cells and in the contextof HCV infection. FIG. 2(A) Schematics of the PCAs format. A and Brepresent prey and bait proteins, and GLuc1/2 are fragments of Gaussialuciferase. FIG. 2(B) Cells were cotransfected with combinations ofplasmids indicated below the graph with those indicated in the legend. Yaxis represents luminescence relative to the core-AP2M1 signal. Thebanded bar on the right represents AP2M1 binding to the host cargoprotein TFR. FIG. 2(C) Core-AP2M1 binding in the presence of free AP2M1,core or NESI. Y axis represents luminescence ratio (the averageluminescence signal detected in cells transfected with Gluc1-AP2M1 andGluc2-core divided by the average of luminescence measured in controlwells transfected with Gluc1-AP2M1 and an empty Gluc2 vector with thosetransfected with Gluc2-core and an empty Gluc1 vector relative tocore-AP2M1 binding in the presence of empty PUC19. FIG. 2(D)Immunoprecipitations (IPs) in membranes of HCV infected cells. Leftpanels: Anti-AP2M1 antibodies or IgG were used for IP. Membranes wereimmunoblotted with anti-phospho-AP2M1, anti-AP2M1, anti-core, andanti-actin antibodies. Cal-A represents calyculin-A. Right panels:Anti-core antibodies or IgG were used for IP. Membranes wereimmunoblotted with anti-core, anti-AP2M1, and anti-actin antibodies.FIG. 2(E) Representative confocal IF microscopy images of AP2M1 and corein Huh-7.5 cells 3 days postelectroporation with J6/JFH HCV RNA. Datarepresent means±s.d. (error bars) from three independent experiments intriplicates (n>20 in E). *p<0.05, **p<0.01, ***p<0.001.

FIG. 3A-H: Shows a core YXXΦ motif mediating AP2M1 binding and viralassembly, functionally interchangeable with other YXXΦ signals. FIG.3(A) Binding of wild type (WT) and core mutants to AP2M1 by PCAs. Y axisrepresents luminescence ratio (the average luminescence signal detectedin cells transfected with Gluc1-AP2M1 and the various forms ofGluc2-core divided by the average of luminescence measured in controlwells transfected with Gluc1-AP2M1 and an empty Gluc2 vector with thosetransfected with the respective Gluc2-core and an empty Gluc1 vector)relative to WT core-AP2M1 binding. FIG. 3(B) HCV RNA replication byRenilla luciferase assays at 6 hr (white) and 72 hr (black)postelectroporation with J6/JFH(p7-Rluc2A) harboring the correspondingmutations. ΔE1-E2 is an assembly defective control. GNN is a replicationdefective polymerase mutant. FIG. 3(C) Extracellular infectivity byluciferase assays in naive Huh-7.5 cells infected with supernatantsderived from the electroporated cells. FIG. 3(D) Intracellularinfectivity by luciferase assays in naive Huh-7.5 cells infected withclarified cell lysates derived from the electroporated cells. FIG. 3(E)Intra- and extracellular infectivity titers measured by limitingdilution assays. TCID₅₀ is a 50% tissue culture infectious dose. FIG.3(F) Viral RNA release into the culture supernatant at 72 hrpostelectroporation measured by qRT-PCR. FIG. 3(G) HCV core proteinrelease into the culture supernatant at 72 hr postelectroporationdetermined by ELISA relative to WT control. FIG. 3(H) Levels of coreprotein by western analysis in lysates prepared from cells infected withvirus harboring the corresponding mutations. Means and s.d. (error bars)of results from at least three independent experiments in triplicatesare shown. The dashed horizontal lines represent background levels ofluciferase activity. RLU is relative light units. *p<0.05, **p<0.01,***p<0.001.

FIG. 4A-I: Shows inhibition of HCV assembly by AP2M1 depletion. FIG.4(A) AP2M1 protein levels by quantitative western analysis in stableclones harboring shRNA lentiviral constructs targeting the AP2M1 geneand a non-targeting (NT) sequence. A representative membrane andcombined data from three independent measurements are shown. Y axisrepresents AP2M1 to actin protein ratio relative to NT control. FIG.4(B) AP2M1/S18 RNA ratio by qRT-PCR in selected stable clones relativeto NT control. FIG. 4(C) The indicated clones were electroporated withJ6/JFH(p7-Rluc2A). HCV RNA replication in these clones by luciferaseassays at 9 hr (white) and 72 hr (black) postelectroporation. FIG. 4(D)Extracellular infectivity measured by luciferase assays in naive cellsinoculated with supernatants derived from the various stable cellclones. FIG. 4(E) Intracellular infectivity by luciferase assays innaive Huh-7.5 cells infected with clarified cell lysates derived fromthe electroporated cells. FIG. 4(F) Intra- and extracellular infectivitytiters measured by limiting dilution assays. TCID₅₀ is a 50% tissueculture infectious dose. FIG. 4(G) Viral RNA release into the culturesupernatant at 72 hr postelectroporation measured by qRT-PCR. FIG. 4(H)HCV core protein release into the culture supernatant at 72 hrpostelectroporation, as determined by ELISA. FIG. 4 (I) Infectious virusproduction relative to NT control (top panel) and levels of AP2M1protein by a western blot analysis (bottom panel) in cells concurrentlytransduced with lentiviruses expressing shAP2M1 and shRNA resistant WTAP2M1 cDNA (AP2M1-WT). Means and s.d. (error bars) of results from atleast three independent experiments are shown. RLU is relative lightunits. *p<0.05, **p<0.01, ***p<0.001.

FIG. 5A-P: Shows that disruption of core-AP2M1 binding abolishesrecruitment of AP2M1 to LD and alters the sub-cellular localization ofcore and its colocalization with E2. Quantitative confocalimmunofluorescence (IF) analysis of the sub-cellular localization ofcore and AP2M1 and core-E2 colocalization in Huh-7.5 cells. FIG. 5(A) Arepresentative merged image of endogenous AP2M1 (blue) and the LDmarker, Bodipy (green), in naive Huh-7.5 cells. FIG. 5(B)-(D) Mergedimages of Huh-7.5 cells infected with J6/JFH HCV stained for core (red),the LD marker, Bodipy (green), and AP2M1 (blue). FIG. 5(E) Percentcolocalization of the indicated signals in naive (white) or infected(black) cells by a quantitative colocalization analysis of (A)-(D). FIG.5(F) A four channel merged image. The yellow arrows in the insetindicate colocalization of core and AP2M1 to LD. FIG. 5(G)-(J)Representative merged images and quantitative colocalization analysis ofAP2M1 (red) and the lipid marker, LipidTOX (blue), in Huh-7.5 cellstransfected with plasmids expressing AP2M1-mCherry alone FIG. 5(G) orAP2M1-mCherry with WT core FIG. 5(H) or core Y136A mutant FIG. 5(I).Core expression (green) in the cells shown in panels FIG. 5(H) and FIG.5(I) is demonstrated in the respective bottom panels. FIG. 5(K)-(P)Representative merged images and quantitative colocalization analysis ofcore (red) and (K) Bodipy (green), demonstrating increased localizationof core to LD in Huh-7.5 cells electroporated with J6/JFH HCV RNAharboring the Y136A core mutation (right panel) compared with WT core(left panel). FIG. 5(L) Bodipy (green) in control (NT) cells (leftpanel) or AP2M1 depleted (right panel) Huh-7.5 electroporated withJ6/JFH HCV RNA, showing a dramatic localization of core to LD in AP2M1depleted cells. FIG. 5(M). TGN46 (green), demonstrating decreasedlocalization of core to TGN in Huh-7.5 cells electroporated with J6/JFHRNA harboring the Y136A core mutation (right panel) compared with WTcore (left panel). FIG. 5(N) TGN46 (green) in control (NT) cells (leftpanel) or AP2M1 depleted (right panel) Huh-7.5 electroporated withJ6/JFH HCV RNA, showing decreased localization of core to TGN in AP2M1depleted cells. FIG. 5(O) E2 (green), demonstrating decreasedcolocalization of core and E2 in Huh-7.5 cells electroporated withJ6/JFH RNA harboring the Y136A core mutation (right panel) compared withWT core (left panel). FIG. 5(P) E2 (green) in control (NT) cells (leftpanel) or AP2M1 depleted (right panel) Huh-7.5 electroporated withJ6/JFH HCV RNA, showing decreased colocalization of core and E2 in AP2M1depleted cells. Representative images at ×60 magnification are shown.Graphs represent quantitative colocalization analysis of Z stacks usingManders' coefficients. Values indicate mean M2 values represented aspercent colocalization (the fraction of green intensity that coincideswith red intensity or in the case of FIGS. FIG. 5(G)-(I), the fractionof blue intensity that coincides with red intensity)±s.d. (error bars);n=10-15. *p<0.05, **p<0.01, ***p<0.001.

FIG. 6A-L: Shows that AAK1 and GAK regulate core-AP2M1 binding and areinvolved in HCV assembly. FIG. 6(A) Regulatory mechanisms of AP2M1binding to host cargo proteins harboring YXXΦ signals. FIG. 6(B) Bindingof core to wild type and T156A AP2M1 mutant by PCAs (black) andmicrofluidics (white). FIG. 6(C)-(E) Huh-7.5 were transfected withplasmids encoding WT or T156A AP2M1 mutant and electroporated withJ6/JFH(p7-Rluc2A) 48 hr posttransfection. FIG. 6(C) Cellular viabilityby alamarBlue-based assays at 48 hr posttransfection relative to WTAP2M1 control. FIG. 6(D) HCV RNA replication in cells overexpressing WTor T156A AP2M1 mutant by luciferase assays at 6 hr (black) and 72 hr(white) postelectroporation with J6/JFH(p7-Rluc2A). FIG. 6(E)Extracellular (black) and intracellular (white) infectivity byluciferase assays in naive Huh-7.5 cells infected with supernatants orcell lysates derived from the indicated cells, respectively, relative toWT control. FIG. 6(F)-(G) Huh7.5 cells were transfected with thecorresponding siRNAs. FIG. 6(F) Ratio of AAK1 (left) or GAK (right) toS18 RNA in these cells relative to NT sequences by qRT-PCR. FIG. 6(G)Quantitative western analysis. Numbers represent AAK1 (top) or GAK(bottom) to actin protein ratios relative to NT control. FIG. 6(H)Core-AP2M1 binding by PCAs in Huh-7.5 cells depleted for AAK1 or GAK bysiRNAs. Y axis represents luminescence ratio (the average luminescencesignal detected in cells transfected with Gluc1-AP2M1 and Gluc2-coredivided by the average of luminescence measured in NT cells transfectedwith Gluc1-AP2M1 and an empty Gluc2 vector with those transfected withGluc2-core and an empty Gluc1 vector) relative to NT control. FIG.6(I)-(K) AAK1 or GAK depleted cells were electroporated withJ6/JFH(p7-Rluc2A). FIG. 6(I) Cellular viability by alamarBlue-basedassays in depleted cells relative to NT control. FIG. 6(J) HCV RNAreplication in these cells by luciferase assays at 6 hr (black) and 72hr (white) postelectroporation. FIG. 6(K) Extracellular (black) andintracellular (white) infectivity by luciferase assays in naive Huh-7.5cells infected with supernatants or cell lysates derived from theindicated cells, respectively, relative to NT control. FIG. 6(L) Corebinding to AAK1 and GAK by PCAs. Y axis represents luminescence ratiorelative to core-AP2M1 binding. Data represent means and s.d. (errorbars) from at least two experiments in triplicates. RLU is relativelight units. *p<0.05, **p<0.01, ***p<0.001.

FIG. 7A-I: Pharmacological inhibition of core-AP2M1 binding and HCVassembly. FIG. 7(A) AP2M1 regulators and the discovered inhibitors. FIG.7(B) The inhibitors' Kds of binding to AAK1 or GAK (Karaman et al., NatBiotechnol 26:127-132, 2008). IC50s for these compounds effect oncore-AP2M1 binding, and EC50s for their effect on extracellularinfectivity, intracellular infectivity, and viral infection with cellculture grown HCV (HCVcc). FIG. 7(C) Inhibition of core-AP2M1 binding bythe compounds measured by PCAs. FIG. 7(D)-(G) Huh-7.5 cellselectroporated with J6/JFH(p7-Rluc2A) were treated daily with eithererlotinib, sunitinib or PKC-412 for 3 days. Supernatants and celllysates were harvested at 72 hr and used to inoculate naive Huh-7.5cells. Dose response curves of the inhibitors' effects on extracellular(D) and intracellular (E) infectivity relative to untreated controls.These compounds had no effect on HCV RNA replication (F) or cellularviability (G) by luciferase and AlamarBlue-based assays, respectively(GNN is a replication-defective polymerase mutant). FIG. 7(H) The effectof the inhibitors on AP2M1 phosphorylation by western analysis of celllysates harvested following electroporation with J6/JFH(p7-Rluc2A) andtreatment with the compounds in the presence of Calyculin A (Cal-A).Representative membranes blotted with anti-phopho-AP2M1 (p-AP2M1) andanti-actin antibodies, and quantitative analysis from 3 experiments areshown. Y axis represents pAP2M1/actin protein ratio relative tountreated controls. FIG. 7(I) The inhibitors' effect on viral infection(black) and cellular viability (grey) in cells infected with HCVccfollowing 72 hr of daily treatment relative to untreated controls. Datarepresent means and s.d. (error bars) from at least three experiments intriplicates. RLU is relative light units. *p<0.05, **p<0.01, ***p<0.001.

FIG. 8A-G: Transient depletion of AP2M1 by pooled siRNAs inhibits HCVassembly. FIG. 8(A) AP2M1/S18 RNA ratio measured by qRT-PCR in Huh-7.5cells transfected with a pool of four siRNAs (ON-TARGETplus SMARTpools,Dharmacon) targeting AP2M1 or a pool of non-targeting (NT) sequences at48 hr posttransfection relative to NT contols. FIG. 8(B) AP2M1 proteinlevels by quantitative western analysis in cells 48 hr posttransfectionwith the corresponding pooled siRNAs. Numbers represent AP2M1 to actinprotein ratio relative to the NT control. FIG. 8(C) Cellular viabilityby alamarBlue assays 48 hr post siRNAs transfections relative to NTcontrol. FIG. 8(D) Cells were electroporated with J6/JFH(p7-Rluc2A) at48 hr following transfection with the indicated pooled siRNAs. HCV RNAreplication in these cells by luciferase assays at 6 hr (black) and 72hr (white) postelectroporation. FIG. 8(E) Extracellular (black) andintracellular (white) infectivity measured in naive Huh-7.5 cellsinfected with supernatants or clarified cell lysates derived fromelectroporated cells harboring the indicated siRNAs by luciferaseassays, respectively. FIG. 8(F) Infectious virus production measured bylimiting dilution assays. FIG. 8(G) Extracellular (black) andintracellular (white) infectivity measured by focus formation assays innaive Huh-7.5 cells infected with supernatants or clarified cell lysatesderived from Huh-7.5 cells transiently depleted for AP2M1 by siRNAs andinfected with culture grown J6/JFH virus (titer: 1.2×10⁵ TCID₅₀/ml).Results are relative to NT controls. Means±s.d. (error bars) of resultsfrom at least two independent experiments are shown. RLU is relativelight units. TCID₅₀ is 50% tissue culture infectious dose.

FIG. 9A-B: Shows that AAK1 and GAK depletion or pharmacologicalinhibition abrogates HIV-1 replication in TZM-b1 cells (CXCR4-positiveHeLa cells that express CD4 and CCR5 and also contain integratedreporter genes for luciferase and E. coli β-galactosidase, both underthe control of an HIV long-terminal repeat sequence (tat gene)). FIG.9(A) HIV replication in AAK1 or GAK depleted cells relative to NTcontrol. FIG. 9 (B) The antiviral effect of AAK1 and GAK inhibitors onHIV replication relative to untreated control.

FIG. 10A-C: Shows Microfluidic-based protein-protein binding assay.Three individual unit cells (out of hundreds in a microfluidic device)are shown in this scheme. FIG. 10(A) Compartments and micromechanicalvalves. A valve is created where a control channel crosses a flowchannel. The resulting thin membrane in the junction between the twochannels can be deflected by hydraulic actuation. Using multiplexedvalve systems allows a large number of elastomeric microvalves toperform complex fluidic manipulations within these devices. FIG. 10(B)Experimental protocol.

represents surface bound biotinylated anti-histidine antibodies (shownin B1 and B2).

represents surface bound FITC-labeled bait human protein (shown in B3).

represents Cy3-labeled prey viral protein (shown in B4 and B5). 1) Themicrofluidic device was bonded to a glass slide and subjected to surfacepatterning that resulted in a circular area coated with biotinylatedanti-histidine antibodies within each unit cell (see c). 2)V5-his-tagged bait human proteins were expressed off the chip using invitro transcription/translation (TNT) mixture and were loaded into thedevice. 3) These proteins bound to the surface anti-his antibodies. 4)T7-tagged viral proteins were expressed off the chip by the samemammalian in vitro TNT mixture in the presence of microsomal membranesand loaded into the device along with FITC-labeled anti-V5 andCy3-labeled anti-T7 antibodies. 5) The “sandwich valves” were closed toallow incubation of the viral protein with the human proteins and theirlabeling with the respective fluorescent antibodies. 6) MITOMI was thenperformed by actuation of the “button membrane” facilitating trappingsurface-bound complexes while expelling any solution phase molecules.The “sandwich valves” were opened followed by a brief wash to removeuntrapped unbound material. 7) The device was scanned by an arrayscanner. Trapped viral protein and surface bound human proteins weredetected. The ratio of bound viral protein to expressed human proteinwas calculated for each data point by measuring the median signal of Cy3to median signal of FITC (represented by

, shown in B7). FIG. 10(C) Surface patterning. 1) Accessible surfacearea was derivatized by flowing a solution of biotinylated BSA (

) through all flow channels. 2) A Neutravidin solution (

) was loaded. 3) The “button” membrane was activated. 4) All remainingaccessible surface area except for a circular area of 60 μm masked bythe button was passivated with biotinylated solution (

). 5) The “button” membrane was opened. 6) A solution ofbiotinylated-anti-his antibodies (

) was loaded allowing specific functionalization of the previouslymasked circular area 7) In vitro expressed human protein (

) bound to the biotinylated-antibodies coating the discrete circulararea. Each of the described steps was followed by a PBS wash.

FIG. 11: Shows that the core's YXXΦ mutations do not affect HCV RNAreplication by qRT-PCR assays. HCV RNA replication, by qRT-PCR inHuh-7.5 cells 72 hr following electroporation with J6/JFH(p7-Rluc2A)harboring the corresponding core's YXXΦ mutations relative to WTcontrol. ΔE1-E2 is an assembly defective control. GNN is a replicationdefective polymerase mutant. Means and s.d. (error bars) of results fromtwo independent experiments in triplicates are shown.

FIG. 12: Shows that AP2M1 depletion has no effect on HCV RNA replicationby qRT-PCR assays. HCV RNA replication, by qRT-PCR in Huh-7.5 cellsharboring the corresponding shRNAs 72 hr following electroporation withJ6/JFH(p7-Rluc2A) relative to NT control. Means and s.d. (error bars) ofresults from two independent experiments in triplicates are shown.

FIG. 13A-G: Shows transient depletion of AP2M1 by pooled siRNAs inhibitsHCV assembly. FIG. 13(A) AP2M1/S18 RNA ratio measured by qRT-PCR inHuh-7.5 cells transfected with a pool of four siRNAs (ON-TARGETplusSMARTpools, Dharmacon) targeting AP2M1 or a pool of non-targeting (NT)sequences at 48 hr posttransfection relative to NT contols. FIG. 13(B)AP2M1 protein levels by quantitative Western analysis in cells 48 hrposttransfection with the corresponding pooled siRNAs. Numbers representAP2M1 to actin protein ratio relative to the NT control. FIG. 13(C)Cellular viability by alamarBlue assays 48 hr post siRNAs transfectionsrelative to NT control. FIG. 13(D) Cells were electroporated withJ6/JFH(p7-Rluc2A) at 48 hr following transfection with the indicatedpooled siRNAs. HCV RNA replication in these cells by luciferase assaysat 6 hr (black) and 72 hr (white) postelectroporation. FIG. 13(E)Extracellular (black) and intracellular (white) infectivity measured innaive Huh-7.5 cells infected with supernatants or clarified cell lysatesderived from electroporated cells harboring the indicated siRNAs byluciferase assays, respectively. FIG. 13(F) Infectious virus productionmeasured by limiting dilution assays. FIG. 13(G) Extracellular (black)and intracellular (white) infectivity measured by focus formation assaysin naive Huh-7.5 cells infected with supernatants or clarified celllysates derived from Huh-7.5 cells transiently depleted for AP2M1 bysiRNAs and infected with culture grown J6/JFH virus (titer: 1.2×10⁵TCID₅₀/ml ). Results are relative to NT controls. Means±s.d. (errorbars) of results from at least two independent experiments are shown.RLU is relative light units. TCID₅₀ is 50% tissue culture infectiousdose.

FIG. 14A-C: Shows that a Y136A core mutation does not seem to affectcore localization to ER membranes in HCV infected cells. FIG. 14(A)quantitative confocal immunofluorescence (IF) analysis for localizationof core to the ER membrane in Huh-7.5 cells infected with culture grownHCV. FIG. 14(A) and FIG. 14(B) are representative merged images of core(red) and the ER marker, calreticulin (green), demonstrating acomparable partial localization of core to the ER membrane in Huh-7.5cells infected with WT virus (A) or with virus harboring the Y136A coremutation (B). Representative images at ×60 magnification are shown. FIG.14(C) Colocalization analysis of Z stacks using Manders' coefficients(with a higher value representing more colocalization). Values indicatemean M2 values represented as percent colocalization (the fraction ofgreen intensity that coincides with red intensity±s.d. (error bars);n=10-15.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and the embodiment of the invention as such may,of course, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to be limiting, since the scope of the presentdisclosure will be limited only by the appended claims.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Before the embodiments of the present disclosure are described indetail, it is to be understood that, unless otherwise indicated, thepresent disclosure is not limited to particular materials, reagents,reaction materials, manufacturing processes, or the like, as such canvary. It is also to be understood that the terminology used herein isfor purposes of describing particular embodiments only, and is notintended to be limiting. It is also possible in the present disclosurethat steps can be executed in different sequence where this is logicallypossible. As used in the specification and the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acompound” includes a plurality of compounds. In this specification andin the claims that follow, reference will be made to a number of termsthat shall be defined to have the following meanings unless a contraryintention is apparent.

Definitions

In describing and claiming the disclosed subject matter, the followingterminology will be used in accordance with the definitions set forthbelow.

By “Flaviviridae virus” is meant any virus of the Flaviviridae family,including those viruses that infect humans and non-human animals. Thepolynucleotide and polypeptides sequences encoding these viruses arewell known in the art, and may be found at NCBI's GenBank database,e.g., as Genbank Accession numbers NC_004102, AB031663, D11355, D11168,AJ238800, NC_001809, NC_001437, NC_004355, NC_004119, NC_003996,NC_003690, NC_003687, NC_003675, NC_003676, NC_003218, NC_001563,NC_000943, NC_003679, NC_003678, NC_003677, NC_002657, NC_002032, andNC_001461, the contents of which database entries are incorporated byreferences herein in their entirety.

As used herein, the terms “treatment,” “treating,” and “treat” aredefined as acting upon a disease, disorder, or condition with an agentto reduce or ameliorate the pharmacologic and/or physiologic effects ofthe disease, disorder, or condition and/or its symptoms. “Treatment,” asused herein, covers any treatment of a disease in a host (e.g., amammal, typically a human or non-human animal of veterinary interest),and includes: (a) reducing the risk of occurrence of the disease in asubject determined to be predisposed to the disease but not yetdiagnosed as infected with the disease, (b) impeding the development ofthe disease, and (c) relieving the disease, i.e., causing regression ofthe disease and/or relieving one or more disease symptoms. “Treatment”is also meant to encompass delivery of an inhibiting agent to provide apharmacologic effect, even in the absence of a disease or condition. Forexample, “treatment” encompasses delivery of a disease or pathogeninhibiting agent that provides for enhanced or desirable effects in thesubject (e.g., reduction of pathogen load, reduction of diseasesymptoms, etc.).

As used herein, the terms “prophylactically treat” or “prophylacticallytreating” refer to completely or partially preventing a disease orsymptom thereof and/or may be therapeutic in terms of a partial orcomplete cure for a disease and/or adverse effect attributable to thedisease.

As used herein, the term “host,” “subject,” “patient,” or “organism”includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, andhorses). Typical hosts to which compounds of the present disclosure maybe administered will be mammals, particularly primates, especiallyhumans. For veterinary applications, a wide variety of subjects will besuitable, e.g., livestock such as cattle, sheep, goats, cows, swine, andthe like; poultry such as chickens, ducks, geese, turkeys, and the like;and domesticated animals, particularly pets such as dogs and cats. Fordiagnostic or research applications, a wide variety of mammals may besuitable subjects, including rodents (e.g., mice, rats, hamsters),rabbits, primates, and swine, such as inbred pigs and the like. The term“living host” refers to a host as noted above or another organism thatis alive. The term “living host” refers to the entire host or organismand not just a part excised (e.g., a liver or other organ) from theliving host.

The term “isolated compound” means a compound which has beensubstantially separated from, or enriched relative to, other compoundswith which it occurs in nature. Isolated compounds are usually at leastabout 80%, at least 90% pure, at least 98% pure, or at least about 99%pure, by weight. The present disclosure is meant to includediastereomers, as well as their racemic and resolved, enantiomericallypure forms and pharmaceutically acceptable salts thereof.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and/or animalsubjects, each unit containing a predetermined quantity of a compound(e.g., an antiviral compound, as described herein) calculated in anamount sufficient to produce the desired effect in association with apharmaceutically acceptable diluent, carrier, or vehicle. Thespecifications for unit dosage forms depend on the particular compoundemployed, the route and frequency of administration, the effect to beachieved, and the pharmacodynamics associated with each compound in thehost.

A “pharmaceutically acceptable excipient,” “pharmaceutically acceptablediluent,” “pharmaceutically acceptable carrier,” or “pharmaceuticallyacceptable adjuvant” means an excipient, diluent, carrier, and/oradjuvant that is useful in preparing a pharmaceutical composition thatis generally safe, non-toxic, and neither biologically nor otherwiseundesirable, and includes an excipient, diluent, carrier, and adjuvantthat are acceptable for veterinary use and/or human pharmaceutical use.“A pharmaceutically acceptable excipient, diluent, carrier and/oradjuvant” as used in the specification and claims includes one and moresuch excipients, diluents, carriers, and adjuvants.

As used herein, a “pharmaceutical composition” is meant to encompass acomposition suitable for administration to a subject, such as a mammal,especially a human. In general a “pharmaceutical composition” issterile, and preferably free of contaminants that are capable ofeliciting an undesirable response within the subject (e.g., thecompound(s) in the pharmaceutical composition is pharmaceutical grade).Pharmaceutical compositions can be designed for administration tosubjects or patients in need thereof via a number of different routes ofadministration including oral, intravenous, buccal, rectal, parenteral,intraperitoneal, intradermal, intracheal, intramuscular, subcutaneous,inhalational, and the like.

The term “therapeutically effective amount” as used herein refers tothat amount of an embodiment of the agent (which may be referred to as acompound, an inhibitory agent, and/or a drug) being administered thatwill relieve to some extent one or more of the symptoms of the disease,i.e., infection, being treated, and/or that amount that will prevent, tosome extent, one or more of the symptoms of the disease, i.e.,infection, that the host being treated has or is at risk of developing.

Viruses are small intracellular infectious agents that replicate via useof the host cell machinery. Virus assembly and budding of Flaviviridaeremain poorly understood, although recent data suggest that theendocytic pathway may be involved, such as with Hepatitis C Virus (HCV).Host proteins are involved in these processes, although prior to thepresent disclosure, host proteins involved in mediating late assemblyand budding were not targeted as antiviral agents. The presentdisclosure, however, provides antiviral methods and compositions thattarget host proteins for late assembly and budding. In particular, hostclathrin adaptor proteins and their regulators involved in the endocyticor secretory pathways are effective targets. The role of clathrinadaptor proteins and their regulators in infectious Flaviviridaeproduction was previously unknown. The present disclosure demonstratesthat host clathrin adaptor proteins, such as AP2M1, AP1M1, AP3M1, andAP4M1, and their regulatoryo proteins, such as, but not limited to AAK1,and GAK are suitable targets for antiviral therapies. In addition, thepresent disclosure provides a novel viral amino acid motif that mediatesinteraction with a host protein.

A conserved YXXΦ motif was discovered within the core protein of HCV, a191-amino acid membrane protein that forms the viral capsid (FIG. 1).This YXXΦ motif is conserved across all HCV isolates available indatabases to date. This motif was also discovered in structural proteinsfrom other Flaviviridae and other viruses. This YXXΦ motif conforms withthe YXXΦ consensus sorting signal within membrane host cargo proteins,recognized by the μ subunit of clathrin adaptor protein (AP) complexes(FIG. 1a ). AP2M1 mediates clathrin-dependent endocytosis, the processby which cargo proteins are sorted into clathrin-coated pits destinedfor fusion with early endosomes. Recognition of the YXXΦ or dileucinemotifs within retroviral proteins by AP2M1 or AP1M1 has been shown to beinvolved in mediating assembly and budding of retroviruses (FIG. 1b )(Batonick et al., Virology 342:190-200, 2005; Camus et al., Molec BiolCell 18:3193-3203, 2007; Berlioz-Torrent et al., J Virol 73:1350-1361,1999; Byland et al., Molec Biol Cell 18:414-425, 2007; Wyss et al., JVirol 75:2982-2992, 2001; Ohno et al., Virology 238:305-315, 1997; Bogeet al., J Biol Chem 273:15773-15778, 1998; Egan et al., J Virol70:6547-6556, 1996; Rowell et al., J Immunol 155:473-488, 1995; Lodge etal., EMBO J 16:695-705, 1997; Deschambeault et al., J Virol73:5010-5017, 1999). However, the role of AAK1 and GAK in HIV infectionhas not been studied, and these mechanisms have not been targetedpharmacologically. Accordingly, the present disclosure provides methodsand compositions for treating or preventing viral infection from HCV,Flaviviridae, Flaviviridae other than HCV, HIV, Lentiviridae other thanHIV, clathrin AP binding viruses, clathrin AP binding other thanFlaviviridae, clathrin AP binding viruses other than HCV, orco-infections such as HCV/HIV co-infections.

Inhibition of the interaction between the viral YXXΦ motif and hostadaptor proteinis, such as, but not limited to AP2M1, results in reducedinfectious virus production of HCV, Flaviviridae, Flaviviridae otherthan HCV, clathrin AP binding viruses, clathrin AP binding viruses otherthan Flaviviridae, clathrin AP binding viruses other than HCV, orco-infections such as HCV/HIV co-infections. In addition, inhibition ofthe activity of proteins that regulate clathrin adaptor proteins mayresult in reduced infectious virus production of HCV, Flaviviridae,Flaviviridae other than HCV, clathrin AP binding viruses, clathrin APbinding viruses other than Flaviviridae, clathrin AP binding virusesother than HCV, or co-infections such as HCV/HIV co-infections. Thus,approaches designed to disrupt the viral YXXΦ motifs interaction withclathrin adaptor proteins, such as AP2M1 and the like, may be useful forinhibiting infectious virus production.

Regulation of the activity of many proteins occurs through the action ofprotein kinases and protein phophatases. GAK and AAK1 are two proteinkinases that modulate the activity of AP2M1 and other μ subunits ofclathrin AP complexes, indicating that administration of protein kinaseinhibitors such as erlotinib, sunitinib, or PKC-412 may abolish theinteraction of AP2M1 and other μ subunits of clathrin AP complexes withviral YXXΦ or dileucine motifs, thereby inhibiting viral infection fromHCV, Flaviviridae, Flaviviridae other than HCV, clathrin AP bindingviruses, clathrin AP binding viruses other than Flaviviridae, clathrinAP binding viruses other than HCV, HIV, Lentiviridae other than HIV, orco-infections such as HCV/HIV co-infections, as described in more detailbelow.

Embodiments of the present disclosure therefore provide methods oftreating a viral infection from clathrin AP binding viruses,Flaviviridae, Lentiviridae, HCV, HIV, co-infections, such as HCV/HIVco-infections, Flaviviridae other than HCV, clathrin AP binding virusesother than Flaviviridae, or clathrin AP binding viruses other than HCV,or any other enveloped virus that hijacks AP2M1 other μ subunits ofclathrin AP complexes/AAK1/GAK, compositions (including inhibitingagents and combinations of such agents with other antiviral therapies)for treating an infection by such viruses, and the like. In particular,embodiments of the present invention provide for methods of treatinginfections caused by clathrin AP binding viruses, Flaviviridae,Lentiviridae, HCV, HIV, co-infections, such as HCV/HIV co-infections,Flaviviridae other than HCV, clathrin AP binding viruses other thanFlaviviridae, or clathrin AP binding viruses other than HCV, andcompositions for treating these infections or any other enveloped virusthat binds clathrin adaptor proteins, such as AP2M1, or is regulated byAAK1 or GAK.

Embodiments of the present disclosure provide methods ofprophylactically treating a viral infection from clathrin AP bindingviruses, Flaviviridae, Lentiviridae, HCV, HIV, co-infections, such asHCV/HIV co-infections, Flaviviridae other than HCV, clathrin AP bindingviruses other than Flaviviridae, or clathrin AP binding viruses otherthan HCV, compositions (including inhibiting agents and combinations ofsuch agents with other antiviral therapies) for prophylacticallytreating an infection by such viruses, and the like. In particular,embodiments of the present invention provide for methods ofprophylactically treating HCV and/or HIV, and compositions for treatingHCV and/or HIV. In an alternative embodiment, the present inventionprovides for methods for treating HCV/HIV infection or Flaviviridaeother than HCV. While the discussion herein may describe the inventionwith respect to HCV, it should be noted that the disclosure relates tonot only HCV, but also to clathrin AP binding viruses, Flaviviridae,Lentiviridae, HIV, co-infections, such as HCV/HIV co-infections,Flaviviridae other than HCV, clathrin AP binding viruses other thanFlaviviridae, or clathrin AP binding viruses other than HCV, andcompositions for treating these infections or any other enveloped virusthat binds AP2M1 or other μ subunits of clathrin AP complexes or isregulated by AAK1 or GAK, or any other enveloped virus that bindsclathrin adaptor proteins, such as AP2M1 and the like, or other μsubunits of clathrin AP complexes and this interaction is regulated byAAK1/GAK.

In view of the recognition that viral proteins comprising a YXXΦ ordileucine motif can mediate interaction with host proteins, embodimentsof the present invention provide compositions (including pharmaceuticalcompositions) including an inhibiting agent that can be used to treat ahost infected by a virus of the Flaviviridae family of viruses. Inparticular, the inhibiting agent can be used to treat hosts infectedwith viral infection from clathrin AP binding viruses, Flaviviridae,Lentiviridae, HCV, HIV, co-infections, such as HCV/HIV co-infections,Flaviviridae other than HCV, clathrin AP binding viruses other thanFlaviviridae, or clathrin AP binding viruses other than HCV, or patientssuffering from coinfections, in particular HCV and HIV or Flaviviridaeother than HCV. Accordingly, two types of inhibiting agents find useherein: (1) those that inhibit interaction between a viral proteincomprising the YXXΦ or dileucine motif and host clathrin adaptorproteins, such as AP2M1 andother μ subunits of clathrin AP complexes;and (2) those that inhibit kinase activity that regulates host proteins.In some embodiments, the inhibiting agent inhibits AAK1 or GAK.

As described in the Examples, it has been observed that the HCV YXXΦmotif in a structural protein binds to the host protein AP2M1. Thisinteraction has been used to identify inhibiting agents that interferewith the interaction and thus may be candidates for clinical developmentas drugs for treating viral infection from clathrin AP binding viruses,Flaviviridae, Lentiviridae, HCV, HIV, co-infections, such as HCV/HIVco-infections, Flaviviridae other than HCV, clathrin AP binding virusesother than Flaviviridae, or clathrin AP binding viruses other than HCV.

Inhibiting agents described herein are useful in the treatment of viralinfections, where the virus is a YXXΦ motif-containing virus, and Y is atyrosine residue, X is any amino acid, and Φ is a bulky hydrophobicresidue. These can include, but are not limited to, phenylalanine,methionine, leucine, isoleucine, and valine. Inhibiting agents describedherein are useful in the treatment of viral infections, where the viruscomprises a dileucine motif-containing virus. Dileucine motifs mayinclude, but are not limited to, leucine-leucine, isoleucine-leucine,leucine-isoleucine, [D/E]XXXL[L/I], or DXXLL (where X is any aminoacid). YXXΦ or dileucine motif-containing viruses include, among others,Lentiviridae, such as HIV, and Flaviviridae family viruses. ExemplaryFlaviviridae include, but are not limited to, flaviviruses,pestiviruses, and hepatitis C viruses. Other YXXΦ or dileucinemotif-containing viruses include yellow fever virus (YFV); Dengue virus,including Dengue types 1-4; Japanese Encephalitis virus; Murray ValleyEncephalitis virus; St. Louis Encephalitis virus; West Nile virus;tick-borne encephalitis virus; Hepatitis C virus (HCV); Kunjin virus;Central European encephalitis virus; Russian spring-summer encephalitisvirus; Powassan virus; Kyasanur Forest disease virus; Ilheus virus; Apoivirus; GB virus A and B; Louping ill virus and Omsk hemorrhagic fevervirus.

In an embodiment, inhibiting agents (e.g., anti-HCV agents) for use ininhibiting HCV replication and treating HCV infection, are of particularinterest. Flaviviridae other than HCV and enveloped viruses other thanFlaviviridae are also of interest. The HCV contemplated by thedisclosure may be of any genotype (genotype 1, 2, 3, 4, 5, 6, and thelike), as well as subtypes of an HCV genotype (e.g., 1a, 1b, 2a, 2b, 3a,etc.). Because HCV genotype 1 is typically the most difficult to treat,the methods and compositions of the invention for treating infections byHCV genotype 1 and genotype 1 subtypes are of particular interest. In anembodiment, HCV co-infections are of interest. In particular, HCV/HIVco-infections are of interest.

While the specification below refers to HCV, such a reference is onlyfor clarity and is not intended to limit the disclosure as described inmore detail below to HCV. As noted above, the methods and compositionsof the invention can be applied to any virus possessing a YXXΦ ordileucine motif in a structural protein (e.g., viral infection from HCV,Flaviviridae, Flaviviridae other than HCV, clathrin AP binding viruses,clathrin AP binding viruses other than Flaviviridae, clathrin AP bindingviruses other than HCV, co-infections such as HCV/HIV co-infections),Lentiviridae or HIV. The compositions and methods can also be applied toco-infections, and infections with Flaviviridae other than HCV, andenveloped viruses other than Flaviviridae.

The instant disclosure also describes in vitro cell-free methods ofidentifying agents (inhibiting agents) that modulate binding between aviral protein containing a YXXΦ or dileucine motif and a host clathrinadaptor protein such as AP2M1 or other μ subunits of clathrin APcomplexes. A test agent that inhibits binding of YXXΦ or dileucinemotif-containing viral proteins to host proteins including clathrinadatpor proteins, such as AP2M1 or other μ subunits of clathrin APcomplexes, can be further tested for its ability to inhibit viralreplication in a cell-based assay. For example, a test agent of interestcan be contacted with a mammalian cell that harbors all or part of anHCV genome, and the effect of the test agent on HCV replication can bedetermined. Suitable cells include mammalian liver cells that arepermissive for HCV replication, e.g., an immortalized human hepatocytecell line that is permissive for HCV. For example, a suitable mammaliancell is Huh7 hepatocyte or a subclone of Huh7 hepatocyte, e.g., Huh-7.5.Suitable cell lines are described in, e.g., Blight et al. (J Virol76:13001, 2002) and Zhang et al. (J Virol 78:1448, 2004). In anembodiment, the HCV genome in the cell comprises a reporter, e.g., anucleotide sequence encoding luciferase, a fluorescent protein, or otherprotein that provides a detectable signal; and determining the effect,if any, of the test agent on HCV replication is achieved by detection ofa signal from the reporter. Other viral assay systems are known in theart.

In one embodiment, the test agents are organic moieties. In thisembodiment, as is generally described in WO 94/24314, which isincorporated herein by reference, test agents are synthesized from aseries of substrates that can be chemically modified. “Chemicallymodified” herein includes traditional chemical reactions, as well asenzymatic reactions. These substrates generally include, but are notlimited to, alkyl groups (including alkanes, alkenes, alkynes, andheteroalkyl), aryl groups (including arenes and heteroaryl), alcohols,ethers, amines, aldehydes, ketones, acids, esters, amides, cycliccompounds, heterocyclic compounds (including purines, pyrimidines,benzodiazepines, beta-lactams, tetracyclines, cephalosporins, andcarbohydrates), steroids (including estrogens, androgens, cortisone,ecodysone, etc.), alkaloids (including ergots, vinca, curare,pyrollizdine, and mitomycines), organometallic compounds, hetero-atombearing compounds, amino acids, and nucleosides. Chemical (includingenzymatic) reactions may be done on the moieties to form new substratesor candidate agents which may then be tested using the present methods.

Thus, in specific embodiments, a test agent of interest (e.g., aninhibiting agent of the invention) inhibits viral replication by atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 80%, or at least about 90%, ormore, compared to the level of HCV replication in the absence of thetest agent.

In particular, embodiments of the present invention include inhibitingagents that inhibit binding of viral proteins containing a YXXΦ ordileucine motif to host clathrin adaptor proteins such as AP2M1 or otherμ subunits of clathrin AP complexes by at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, or at least about 90%, or more, compared to the bindingof viral proteins containing a YXXΦ or dileucine motif to host clathrinadaptor proteins such as AP2M1 or other μ subunits of clathrin APcomplexes in the absence of the test agent.

In yet another embodiment, the inhibiting agent is one that inhibitsbinding of a viral protein containing a YXXΦ or dileucine motif to hostclathrin adaptor proteins such as AP2M1 or other μ subunits of clathrinAP complexes with a 50% inhibitory concentration (IC₅₀) of about 100 μMto 50 μM, about 50 μM to 25 μM, about 25 μM to 10 μM, about 10 μM to 5μM, about 5 μM to 1 μM, about 1 μM to 500 nM, about 500 nM to 400 nM,about 400 nM to 300 nM, about 300 nM to 250 nM, about 250 nM to 200 nM,about 200 nM to 150 nM, about 150 nM to 100 nM, about 100 nM to 50 nM,about 50 nM to 30 nM, about 30 nM to 25 nM, about 25 nM to 20 nM, about20 nM to 15 nM, about 15 nM to 10 nM, about 10 nM to 5 nM, or less thanabout 5 nM.

In still yet another embodiment, the inhibiting agent inhibits bindingof at least one viral protein containing a YXXΦ or dileucine motif tohost clathrin adaptor proteins such as AP2M1 or other μ subunits ofclathrin AP complexes. In an embodiment, the inhibiting agent inhibitsbinding of at least one viral protein containing a YXXΦ or dileucinemotif to host proteins such as AP2M1 or other μ subunits of clathrin APcomplexes with an IC₅₀ of less than about 500 nM, e.g., in someembodiments, the inhibiting agent inhibits binding of at least one viralprotein containing a YXXΦ or dileucine motif to host structural proteinssuch as AP2M1 or other μ subunits of clathrin AP complexes with an IC₅₀of about 500 nM to 400 nM, about 400 nM to 300 nM, about 300 nM to 250nM, about 250 nM to 200 nM, about 200 nM to 150 nM, about 150 nM to 100nM, about 100 nM to 50 nM, about 50 nM to 30 nM, about 30 nM to 25 nM,about 25 nM to 20 nM, about 20 nM to 15 nM, about 15 nM to 10 nM, about10 nM to 5 nM, or less than about 5 nM.

In still yet another embodiment, the inhibiting agent, when contactedwith a virus-infected cell (e.g., an HCV-infected liver cell), inhibitsviral replication in the cell by at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, or at least about 90%, or more, compared to the level ofviral replication in a viral-infected cell not contacted with theinhibiting agent.

In still yet another embodiment, the inhibiting agent, when contactedwith an virus-infected cell (e.g., an HCV-infected liver cell), reducesthe amount of infectious viral particles produced by the infected cellby at least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, or at least about90%, or more, compared to the number of infectious viral particlesproduced by the cell not contacted with the inhibiting agent.

In still yet another embodiment, in addition to determining the effectof a test agent on inhibition of viral proteins containing a YXXΦ ordileucine motif to host clathrin adaptor proteins such as AP2M1 or otherμ subunits of clathrin AP complexes, test agents are assessed for anycytotoxic activity they may exhibit toward a living eukaryotic cell,using well-known assays, such as trypan blue dye exclusion, an MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)assay, and the like. Agents that do not exhibit significant cytotoxicactivity may be considered preferred agents for further development asdrugs.

In still yet another embodiment, the inhibiting agent, when administeredin one or more doses to an individual infected with a virus as describedherein (e.g., a human), reduces the viral load in the individual by atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 80%, or at least about 90%, ormore, compared to the viral load in the individual not treated with theinhibiting agent.

Inhibiting Agents

Embodiments of the present disclosure provide for inhibiting agents thatcan be used to treat a host infected by a virus of the Flaviviridaefamily of viruses. In particular, Flaviviridae other than HCV. Inparticular, the inhibiting agent can be used to treat hosts infectedwith Hepatitis C virus or coinfections, in particular HCV+HIV. Theinhibiting agent can be in one or more of the following groups:inhibitors of protein kinases AAK1 or GAK, which regulate viralprotein-clathrin adaptor, such as AP2M1 (or other μ subunits of clathrinAP complexes), binding and mediate virus assembly and/or budding. SinceAP2M1 and other μ subunits of clathrin AP complexes mediate variousstages in the life cycles of multiple viruses, inhibition of AP2M1 andother μ subunits of clathrin AP complexes, such as by inhibition of itsregulatory protein kinases or phosphatases, may affect or inhibitmultiple viruses. Accordingly, the protein kinases that regulate thesehost proteins may be functionally relevant in other viral families.While not exclusively specific (like most compounds), the discoveredprotein kinase inhibitors bind AAK1 or GAK with high affinities comparedto their other targets and have no effect on HCV RNA replication,demonstrating a relatively good selectivity.

Other agents that find use as inhibitors of GAK and AAK1 include, butare not limited to, RNAi, antisense, ribozymes, or small molecules thatcompete with erlotinib, sunitinib, or PKC-412 for binding to GAK orAAK1. In addition, inhibitors that are able to compete with YXXΦ ordileucine motifs for binding to host proteins include, but are notlimited to, binding agents such as antibodies directed to YXXΦ ordileucine or against AP2M1 or other μ subunits of clathrin AP complexes.In addition, dominant-negative binding proteins or aptamers can inhibitGAK or AAK1. In another embodiment, decoy receptors or polypeptidescorresponding to the YXXΦ or dileucine binding site from AP2M1 other μsubunits of clathrin AP complexes can inhibit GAK or AAK1.

Embodiments of the present invention include salts of the inhibitingagents. Embodiments of the present invention include prodrugs of theinhibiting agents. Embodiments of the invention include in compositions,pharmaceutical compositions, liquid compositions, gel compositions, andthe like, each containing an inhibiting agent identified herein, andeach of these compositions, in an embodiment, can be in the form of acontrolled release or a sustained release formulation. In an embodiment,the inhibiting agent can be used in combination with another agent usedto treat a Flaviviridae family viral infection, and as previously noted,either of the agents (the inhibiting agent and the other agent) or bothof the agents can be in the form of a controlled release or a sustainedrelease. In some instances, the term “inhibiting agent” may be referredto as an active agent or drug.

Pharmaceutical Formulations and Routes of Administration

Embodiments of the present disclosure include one or more inhibitingagents identified herein and formulated with one or morepharmaceutically acceptable excipients, diluents, carriers and/oradjuvants. In addition, embodiments of the present invention includesuch inhibiting agents formulated with one or more pharmaceuticallyacceptable auxiliary substances. In particular, one or more inhibitingagents can be formulated with one or more pharmaceutically acceptableexcipients, diluents, carriers, and/or adjuvants to provide anembodiment of a composition of the invention.

In an embodiment, the inhibiting agent can be combined with anotherantiviral agent to prepare a composition of the invention, and thecomposition can include one or more pharmaceutically acceptableexcipients, diluents, carriers and/or adjuvants.

In an embodiment, an inhibiting agent that inhibits binding of a proteincontaining a YXXΦ or dileucine motif to a host clathrin adaptor proteinsuch as AP2M1 or other subunits of clathrin AP complexes (referred tobelow as “a subject active agent” or “drug”) can be formulated with oneor more pharmaceutically acceptable excipients, diluents, carriers,and/or adjuvants.

A wide variety of pharmaceutically acceptable excipients are known inthe art. Pharmaceutically acceptable excipients have been amplydescribed in a variety of publications, including, for example, Gennaro(2000), “Remington: The Science and Practice of Pharmacy”;Pharmaceutical Dosage Forms and Drug Delivery Systems (1999); andHandbook of Pharmaceutical Excipients (2000).

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents, and the like, are readily available to the public.

In an embodiment of the present disclosure, the inhibiting agent isadministered to the host using any means capable of resulting in thedesired effect (e.g., reduction in viral load, reduction in liverfibrosis, increase in liver function, and the like). Thus, theinhibiting agent can be incorporated into a variety of formulations fortherapeutic administration. For example, the inhibiting agent can beformulated into pharmaceutical compositions by combination withappropriate, pharmaceutically acceptable carriers or diluents, and maybe formulated into preparations in solid, semi-solid, liquid, or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, and aerosols.

In pharmaceutical dosage forms, the inhibiting agent may be administeredin the form of its pharmaceutically acceptable salts, or a subjectactive agent may be used alone or in appropriate association, as well asin combination, with other pharmaceutically active compounds. Thefollowing methods and excipients are merely exemplary and are in no waylimiting.

For oral preparations, the inhibiting agent can be used alone or incombination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch, or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch, orgelatins; with disintegrators, such as corn starch, potato starch, orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives, and flavoring agents.

Embodiments of the inhibiting agent can be formulated into preparationsfor injection by dissolving, suspending, or emulsifying them in anaqueous or nonaqueous solvent, such as vegetable or other similar oils,synthetic aliphatic acid glycerides, esters of higher aliphatic acids,or propylene glycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers, and preservatives.

Embodiments of the inhibiting agent can be utilized in aerosolformulation to be administered via inhalation. Embodiments of theinhibiting agent can be formulated into pressurized acceptablepropellants such as dichlorodifluoromethane, propane, nitrogen and thelike.

Furthermore, embodiments of the inhibiting agent can be made intosuppositories by mixing with a variety of bases such as emulsifyingbases or water-soluble bases. Embodiments of the inhibiting agent can beadministered rectally via a suppository. The suppository can includevehicles such as cocoa butter, carbowaxes, and polyethylene glycols,which melt at body temperature, yet are solidified at room temperature.

Unit dosage forms for oral or rectal administration, such as syrups,elixirs, and suspensions, may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet, or suppository, contains apredetermined amount of the composition containing one or moreinhibiting agents. Similarly, unit dosage forms for injection orintravenous administration may comprise the inhibiting agent in acomposition as a solution in sterile water, normal saline, or anotherpharmaceutically acceptable carrier.

Embodiments of the inhibiting agent can be formulated in an injectablecomposition in accordance with the invention. Typically, injectablecompositions are prepared as liquid solutions or suspensions; solidforms suitable for solution in, or suspension in, liquid vehicles priorto injection may also be prepared. The preparation may also beemulsified or the active ingredient (inhibiting agent) encapsulated inliposome vehicles in accordance with the invention.

In an embodiment, the inhibiting agent is formulated for delivery by acontinuous delivery system. The term “continuous delivery system” isused interchangeably herein with “controlled delivery system” andencompasses continuous (e.g., controlled) delivery devices (e.g., pumps)in combination with catheters, injection devices, and the like, a widevariety of which are known in the art.

Mechanical or electromechanical infusion pumps can also be suitable foruse with the present disclosure. Examples of such devices include thosedescribed in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019;4,487,603; 4,360,019; 4,725,852; 5,820,589; 5,643,207; 6,198,966; andthe like. In general, delivery of the inhibiting agent can beaccomplished using any of a variety of refillable pump systems. Pumpsprovide consistent, controlled release over time. In some embodiments,the inhibiting agent is in a liquid formulation in a drug-impermeablereservoir, and is delivered in a continuous fashion to the individual.

In one embodiment, the drug delivery system is an at least partiallyimplantable device. The implantable device can be implanted at anysuitable implantation site using methods and devices well known in theart. An implantation site is a site within the body of a subject atwhich a drug delivery device is introduced and positioned. Implantationsites include, but are not necessarily limited to, a subdermal,subcutaneous, intramuscular, or other suitable site within a subject'sbody. Subcutaneous implantation sites are used in some embodimentsbecause of convenience in implantation and removal of the drug deliverydevice.

Drug release devices suitable for use in the disclosure may be based onany of a variety of modes of operation. For example, the drug releasedevice can be based upon a diffusive system, a convective system, or anerodible system (e.g., an erosion-based system). For example, the drugrelease device can be an electrochemical pump, osmotic pump, anelectroosmotic pump, a vapor pressure pump, or osmotic bursting matrix,e.g., where the drug is incorporated into a polymer and the polymerprovides for release of drug formulation concomitant with degradation ofa drug-impregnated polymeric material (e.g., a biodegradable,drug-impregnated polymeric material). In other embodiments, the drugrelease device is based upon an electrodiffusion system, an electrolyticpump, an effervescent pump, a piezoelectric pump, a hydrolytic system,etc.

Drug release devices based upon a mechanical or electromechanicalinfusion pump can also be suitable for use with the present disclosure.Examples of such devices include those described in, for example, U.S.Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852, and thelike. In general, a subject treatment method can be accomplished usingany of a variety of refillable, non-exchangeable pump systems. Pumps andother convective systems are generally preferred due to their generallymore consistent, controlled release over time. Osmotic pumps are used insome embodiments due to their combined advantages of more consistentcontrolled release and relatively small size (see, e.g., PCT publishedapplication no. WO 97/27840 and U.S. Pat. Nos. 5,985,305 and 5,728,396).Exemplary osmotically-driven devices suitable for use in the disclosureinclude, but are not necessarily limited to, those described in U.S.Pat. Nos. 3,760,984; 3,845,770; 3,916,899; 3,923,426; 3,987,790;3,995,631; 3,916,899; 4,016,880; 4,036,228; 4,111,202; 4,111,203;4,203,440; 4,203,442; 4,210,139; 4,327,725; 4,627,850; 4,865,845;5,057,318; 5,059,423; 5,112,614; 5,137,727; 5,234,692; 5,234,693;5,728,396; and the like.

In some embodiments, the drug delivery device is an implantable device.The drug delivery device can be implanted at any suitable implantationsite using methods and devices well known in the art. As noted herein,an implantation site is a site within the body of a subject at which adrug delivery device is introduced and positioned. Implantation sitesinclude, but are not necessarily limited to a subdermal, subcutaneous,intramuscular, or other suitable site within a subject's body.

In some embodiments, an active agent is delivered using an implantabledrug delivery system, e.g., a system that is programmable to provide foradministration of the agent. Exemplary programmable, implantable systemsinclude implantable infusion pumps. Exemplary implantable infusionpumps, or devices useful in connection with such pumps, are describedin, for example, U.S. Pat. Nos. 4,350,155; 5,443,450; 5,814,019;5,976,109; 6,017,328; 6,171,276; 6,241,704; 6,464,687; 6,475,180; and6,512,954. A further exemplary device that can be adapted for thepresent disclosure is the Synchromed Infusion pump (Medtronic).

Suitable excipient vehicles for the inhibiting agent are, for example,water, saline, dextrose, glycerol, ethanol, or the like, andcombinations thereof. In addition, if desired, the vehicle may containminor amounts of auxiliary substances such as wetting or emulsifyingagents or pH buffering agents. Methods of preparing such dosage formsare known, or will be apparent upon consideration of this disclosure, tothose skilled in the art. See, e.g., Remington's PharmaceuticalSciences, 17th edition, 1985. The composition or formulation to beadministered will, in any event, contain a quantity of the inhibitingagent adequate to achieve the desired state in the subject beingtreated.

Compositions of the present invention include those that comprise asustained-release or controlled release matrix. In addition, embodimentsof the present invention can be used in conjunction with othertreatments that use sustained-release formulations. As used herein, asustained-release matrix is a matrix made of materials, usuallypolymers, which are degradable by enzymatic or acid-based hydrolysis orby dissolution. Once inserted into the body, the matrix is acted upon byenzymes and body fluids. A sustained-release matrix desirably is chosenfrom biocompatible materials such as liposomes, polylactides (polylacticacid), polyglycolide (polymer of glycolic acid), polylactideco-glycolide (copolymers of lactic acid and glycolic acid),polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid,collagen, chondroitin sulfate, carboxcylic acids, fatty acids,phospholipids, polysaccharides, nucleic acids, polyamino acids, aminoacids such as phenylalanine, tyrosine, isoleucine, polynucleotides,polyvinyl propylene, polyvinylpyrrolidone, and silicone. Illustrativebiodegradable matrices include a polylactide matrix, a polyglycolidematrix, and a polylactide co-glycolide (co-polymers of lactic acid andglycolic acid) matrix.

In another embodiment, the pharmaceutical composition of the presentdisclosure (as well as combination compositions) is delivered in acontrolled release system. For example, the inhibiting agent may beadministered using intravenous infusion, an implantable osmotic pump, atransdermal patch, liposomes, or other modes of administration. In oneembodiment, a pump may be used (Sefton 1987; Buchwald et al. 1980;Saudek et al. 1989). In another embodiment, polymeric materials areused. In yet another embodiment a controlled release system is placed inproximity of the therapeutic target, i.e., the liver, thus requiringonly a fraction of the systemic dose. In yet another embodiment, acontrolled release system is placed in proximity of the therapeutictarget, thus requiring only a fraction of the systemic. Other controlledrelease systems are discussed in the review by Langer (1990).

In another embodiment, the compositions of the present invention (aswell as combination compositions separately or together) include thoseformed by impregnation of an inhibiting agent described herein intoabsorptive materials, such as sutures, bandages, and gauze, or coatedonto the surface of solid phase materials, such as surgical staples,zippers and catheters to deliver the compositions. Other deliverysystems of this type will be readily apparent to those skilled in theart in view of the instant disclosure.

Treatment Methods

Embodiments of the present invention include methods of treating aninfection by viral infection from clathrin AP binding viruses,Flaviviridae, Lentiviridae, HCV, HIV, co-infections, such as HCV/HIVco-infections, Flaviviridae other than HCV, clathrin AP binding virusesother than Flaviviridae, or clathrin AP binding viruses other than HCV.In particular, inhibiting agents described herein can be used to treatan infection by a virus of the Flaviviridae family of viruses. In anembodiment, the present disclosure provides a method of treating a hostinfected with a virus as described above by administering to the host atherapeutically effective amount of an inhibiting agent in one or moredoses, to reduce the viral load in the host.

Embodiments of the present invention include methods of prophylacticallytreating an infection by a viral infection from clathrin AP bindingviruses, Flaviviridae, Lentiviridae, HCV, HIV, co-infections, such asHCV/HIV co-infections, Flaviviridae other than HCV, clathrin AP bindingviruses other than Flaviviridae, or clathrin AP binding viruses otherthan HCV. In particular, inhibiting agents described herein can be usedto prophylactically treat an infection by a virus of the Flaviviridaefamily of viruses. In an embodiment, the present disclosure provides amethod of prophylactically treating a host infected with a virus fromthe Flaviviridae family of viruses by administering to the host atherapeutically effective amount of an inhibiting agent in one or moredoses, to reduce the viral load in the host. In an embodiment, a methodof prophylactically treating a host infected with a virus from theFlaviviridae family of viruses, the method comprising administering tothe host a therapeutically effective amount of an inhibiting agent toreduce the viral load in the host. Additional details regardingclemizole and dosing of clemizole is described above.

In an embodiment, inhibiting agents described herein are used incombination with another agent (e.g., an antiviral agent) to treat aninfection with viral infection from clathrin AP binding viruses,Flaviviridae, Lentiviridae, HCV, HIV, co-infections, such as HCV/HIVco-infections, Flaviviridae other than HCV, clathrin AP binding virusesother than Flaviviridae, or clathrin AP binding viruses other than HCV.In an embodiment, inhibiting agents described herein are used incombination with another agent (e.g., an antiviral agent) toprophylactically treat an infection with a virus from the Flaviviridaefamily of viruses. Embodiments of the method involve administering to anindividual in need thereof one or more inhibiting agents that inhibitbinding of viral proteins containing a YXXΦ or dileucine motif to hostclathrin adaptor proteins such as AP2M1 or other μ subunits of clathrinAP complexes. In an embodiment, the present invention provides methodsof treating a flavivirus infection, e.g., an HCV infection, and methodsof reducing liver fibrosis that may occur as sequelae of an HCVinfection.

In an embodiment, the inhibiting agent includes one or more ofinhibiting agents described above. In various embodiments, theinhibiting agent is a protein kinase inhibitor, such as inhibitors ofAAK1 or GAK, which regulates viral protein-AP2M1 binding or binding of aviral protein to other μ subunits of clathrin AP complexes and mediatevirus assembly and/or budding, or variants of these proteins thereof.

In an embodiment, an effective amount of the inhibiting agent is anamount that, when administered in one or more doses to a host (e.g.,human) in need thereof, reduces viral load in the individual by at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, or at least about 90%, or more, comparedto the viral load in the individual not treated with the inhibitingagent.

Viral load can be measured by measuring the titer or level of virus inserum. These methods include, but are not limited to, a quantitativepolymerase chain reaction (PCR) and a branched DNA (bDNA) test.Quantitative assays for measuring the viral load (titer) of HCV RNA havebeen developed. Many such assays are available commercially, including aquantitative reverse transcription PCR (qRT-PCR) (Amplicor HCV Monitor™,Roche Molecular Systems, New Jersey); and a branched DNA signalamplification assay [Quantiplex™ HCV RNA Assay (bDNA), Chiron Corp.,Emeryville, Calif.]. See, e.g., Gretch et al. (1995). Also of interestis a nucleic acid test (NAT) sold by Chiron Corporation under the tradename Procleix™, which NAT simultaneously tests for the presence of HIV-1and HCV (Vargo et al. 2002).

In some embodiments, an effective amount of the inhibiting agent is anamount that, when administered in one or more doses to a host (e.g.,human) in need thereof, increases liver function in the individual by atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, or at least about 90%, or more,compared to the liver function in the individual not treated with theinhibiting agent.

In some embodiments, an effective amount of the inhibiting agent is anamount that, when administered in one or more doses to a host (e.g., ahuman) in need thereof, reduces liver fibrosis in the host by at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, or at least about 90%, or more, comparedto the degree of liver fibrosis in the individual not treated with theinhibiting agent.

Embodiments of the present disclosure provide methods, inhibitingagents, and pharmaceutical formulations useful in the treatment ofpatients suffering from a viral infection from clathrin AP bindingviruses, Flaviviridae, Lentiviridae, HCV, HIV, co-infections, such asHCV/HIV co-infections, Flaviviridae other than HCV, clathrin AP bindingviruses other than Flaviviridae, or clathrin AP binding viruses otherthan HCV. In one embodiment, the patient is co-infected withFlaviviridae other than HCV. In one embodiment, the patient is infectedwith HCV but is not known to be infected with another virus, including,but not limited to, HIV. In another embodiment, the patient is infectedwith HCV and one or more additional viruses, including, but not limitedto, HIV. In one embodiment, the patient is treated for a viral infectionby administering only a single inhibiting agent as described herein asuseful in the treatment of HCV infection. In another embodiment, thepatient is treated for a viral infection by administering both aninhibiting agent described herein as useful in the treatment of HCVinfection as well as one or more additional agents known to be useful inthe treatment of viral infection, including, but not limited to drugsfor the treatment of HIV, such as Atripla, Complera, Combivir, Retrovir,Truvada, Viracept, Fuzeon, Selzentry, Isentress, or the like. In oneembodiment, the one or more additional agents does not include a CCR-5antagonist. In another embodiment, the one or more additional agentsdoes include a CCR-5 antagonist, and the patient is infected with HCVbut not known to be infected (or is not infected) with HIV.

Dosages

Embodiments of the inhibiting agent can be administered to a host in oneor more doses. In an embodiment, the inhibiting agent can beadministered in an amount of about 10 mg to 1000 mg per dose, e.g.,about 10 mg to 20 mg, about 20 mg to 25 mg, about 25 mg to 50 mg, about50 mg to 75 mg, about 75 mg to 100 mg, about 100 mg to 125 mg, about 125mg to 150 mg, about 150 mg to 175 mg, about 175 mg to 200 mg, about 200mg to 225 mg, about 225 mg to 250 mg, about 250 mg to 300 mg, about 300mg to 350 mg, about 350 mg to 400 mg, about 400 mg to 450 mg, about 450mg to 500 mg, about 500 mg to 750 mg, or about 750 mg to 1000 mg perdose.

In an embodiment, the amount of the inhibiting agent per dose isdetermined on a per body weight basis. For example, in an embodiment,the inhibiting agent can be administered in an amount of about 0.5 mg/kgto 100 mg/kg, e.g., about 0.5 mg/kg to 1 mg/kg, about 1 mg/kg to 2mg/kg, about 2 mg/kg to 3 mg/kg, about 3 mg/kg to 5 mg/kg, about 5 mg/kgto 7 mg/kg, about 7 mg/kg to about 10 mg/kg, about 10 mg/kg to 15 mg/kg,about 15 mg/kg to 20 mg/kg, about 20 mg/kg to 25 mg/kg, about 25 mg/kgto 30 mg/kg, about 30 mg/kg to 40 mg/kg, about 40 mg/kg to 50 mg/kg,about 50 mg/kg to 60 mg/kg, about 60 mg/kg to 70 mg/kg, about 70 mg/kgto 80 mg/kg, about 80 mg/kg to 90 mg/kg, or about 90 mg/kg to 100 mg/kg,or more than about 100 mg/kg.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific inhibiting agent administered, the severity ofthe symptoms and the susceptibility of the subject to side effects.Preferred dosages for a given compound are readily determinable by thoseof skill in the art by a variety of means.

In an embodiment, multiple doses of the inhibiting agent areadministered. The frequency of administration of the inhibiting agentcan vary depending on any of a variety of factors, e.g., severity of thesymptoms, and the like. For example, in an embodiment, the inhibitingagent is administered once per month, twice per month, three times permonth, every other week (qow), once per week (qw), twice per week (biw),three times per week (tiw), four times per week, five times per week,six times per week, every other day (qod), daily (qd), twice a day(qid), or three times a day (tid). As discussed above, in an embodiment,the inhibiting agent is administered continuously.

The duration of administration of the inhibiting agent, e.g., the periodof time over which the inhibiting agent is administered, can vary,depending on any of a variety of factors, e.g., patient response, etc.For example, the inhibiting agent can be administered over a period oftime of about one day to one week, about two weeks to four weeks, aboutone month to two months, about two months to four months, about fourmonths to six months, about six months to eight months, about eightmonths to 1 year, about 1 year to 2 years, or about 2 years to 4 years,or more.

Routes of Administration

Embodiments of the present invention provide methods and compositionsfor the administration of the inhibiting agent to a host (e.g., a human)using any available method and route suitable for drug delivery,including in vivo and ex vivo methods, as well as systemic and localizedroutes of administration.

Routes of administration include intranasal, intramuscular,intratracheal, subcutaneous, intradermal, topical application,intravenous, rectal, nasal, oral, and other enteral and parenteralroutes of administration. Routes of administration may be combined, ifdesired, or adjusted depending upon the agent and/or the desired effect.An active agent can be administered in a single dose or in multipledoses.

Embodiments of the inhibiting agent can be administered to a host usingavailable conventional methods and routes suitable for delivery ofconventional drugs, including systemic or localized routes. In general,routes of administration contemplated by the disclosure include, but arenot limited to, enteral, parenteral, or inhalational routes.

Parenteral routes of administration other than inhalation administrationinclude, but are not limited to, topical, transdermal, subcutaneous,intramuscular, intraorbital, intracapsular, intraspinal, intrasternal,and intravenous routes, i.e., any route of administration other thanthrough the alimentary canal. Parenteral administration can be conductedto effect systemic or local delivery of the inhibiting agent. Wheresystemic delivery is desired, administration typically involves invasiveor systemically absorbed topical or mucosal administration ofpharmaceutical preparations.

The inhibiting agent can also be delivered to the subject by enteraladministration. Enteral routes of administration include, but are notlimited to, oral and rectal (e.g., using a suppository) delivery.

Methods of administration of the inhibiting agent through the skin ormucosa include, but are not limited to, topical application of asuitable pharmaceutical preparation, transdermal transmission, injectionand epidermal administration. For transdermal transmission, absorptionpromoters or iontophoresis are suitable methods. Iontophoretictransmission may be accomplished using commercially available “patches”that deliver their product continuously via electric pulses throughunbroken skin for periods of several days or more.

Combination Therapies

Embodiments of the present invention include methods, inhibiting agents,and pharmaceutical formulations for the treatment of viral infection.Embodiments of the inhibiting agents and pharmaceutical formulationsuseful in the methods of the present disclosure can be employed incombination with other antiviral agents to treat viral infection. In anembodiment, in accordance with the methods of the present invention, aninhibiting agent that is used to treat a host infected by viralinfection from clathrin AP binding viruses, Flaviviridae, Lentiviridae,HCV, HIV, co-infections, such as HCV/HIV co-infections, Flaviviridaeother than HCV, clathrin AP binding viruses other than Flaviviridae, orclathrin AP binding viruses other than HCV is used in combination withone or more other antiviral agents to treat the infection. In anembodiment, in accordance with the methods of the present invention, aninhibiting agent that prevents the binding of at least one viral proteincontaining a YXXΦ or dileucine motif to host proteins such as AP2M1 orother μ subunits of clathrin AP complexes (also referred to herein as an“HCV YXXΦ or dileucine antagonist”) can be used in combination with oneor more other antiviral agents to treat viral infection.

For instance, current medical practice to treat HCV infection typicallyemploys combination therapy with ribavirin (such as Rebetol or Copegus),either an interferon-alpha (such as interferon alpha 2b) or pegylatedinterferon (such as Pegasys, marketed by Roche, or PEG-Intron, marketedby Schering Plough), and a protease inhibitor. In accordance with themethods of the present disclosure, an inhibiting compound can be used incombination with these standard therapies to treat HCV infection.

A number of HCV protease and polymerase inhibitors are either approvedor in development for the treatment of HCV infection, and in accordancewith the methods of the present disclosure, co-administration of aninhibiting agent that prevents the binding of at least one viral proteincontaining a YXXΦ or dileucine motif to host proteins such as AP2M1 orother μ subunits of clathrin AP complexes and an HCV protease inhibitorcan be efficacious in the treatment of HCV. In one embodiment, aninterferon alpha and/or a nucleoside analog such as ribavirin is/arealso employed in this combination therapy. Suitable HCV proteaseinhibitors include, but are not limited to, telaprevir (VX-950, Vertex),BILN 2061 and BI12202 (Boehringer Ingelheim), boceprevir (SCH 503034,Schering Plough), ITMN191 (Roche/InterMune/Array BioPharma), MK-7009(Merck), TMC435350 (Tibotec/Medivir), ACH-1095 and ACH-806(Achillion/Gilead), and other inhibitors of NS3/NS4A protease,including, but not limited to, compounds in development by Presidio.

In accordance with the methods of the present disclosure,co-administration of an inhibiting agent that prevents the binding of atleast one viral protein containing a YXXΦ or dileucine motif to hostproteins such as AP2M1 or other μ subunits of clathrin AP complexes andan HCV RNA polymerase inhibitor can be efficacious in the treatment ofHCV. In one embodiment, an interferon alpha and/or a nucleoside analogsuch as ribavirin and/or an HCV protease inhibitor is/are also employedin this combination therapy. Suitable HCV RNA polymerase inhibitorsinclude, but are not limited to, valopicitabine (NM283,Idenix/Novartis), HCV-796 (Wyeth/ViroPharma), R1626 (Roche), R7128(Roche/Pharmasset), GS-9190 (Gilead), MK-0608 (Merck), PSI-6130(Pharmasset), and PFE-868,554 (PFE). In an embodiment, the methodprovides combination treatments with agents that inhibit AAK1 and GAK.

A number of toll-like receptor (TLR) agonists are in development for thetreatment of HCV infection, and in accordance with the methods of thepresent disclosure, co-administration of a YXXΦ or dileucine antagonistthat prevents the binding of at least one viral protein containing aYXXΦ or dileucine motif to host proteins such as AP2M1 or other μsubunits of clathrin AP complexes and a TLR agonist can be efficaciousin the treatment of HCV. In one embodiment, an interferon alpha and/or anucleoside analog such as ribavirin and/or an HCV protease inhibitorand/or an HCV RNA polymerase inhibitor is/are also employed in thiscombination therapy. Suitable TLR agonists include, but are not limitedto, TLR7 agonists [i.e., ANA245 and ANA975 (Anadys/Novartis)] and TLR9agonists [i.e., Actilon (Coley) and IMO-2125 (Idera)].

A number of thiazolide derivatives are in development for the treatmentof HCV infection, and in accordance with the methods of the presentdisclosure, co-administration of an antagonist that prevents the bindingof at least one viral protein containing a YXXΦ or dileucine motif tohost proteins such as AP2M1 or other μ subunits of clathrin APcomplexes, and a thiazolide, including, but not limited to, Nitazoxanide(Alinia, or other sustained release formulations of nitazoxanide orother thiazolides, Romark Laboratories) can be efficacious in thetreatment of HCV. In an embodiment, an interferon alpha and/or anucleoside analog such as ribavirin and/or an HCV protease inhibitorand/or an HCV RNA polymerase inhibitor and/or a TLR agonist is/are alsoemployed in this combination therapy.

In another embodiment of the methods of the present disclosure,co-administration of an inhibiting agent that prevents the binding of atleast one viral protein containing a YXXΦ or dileucine motif to hostproteins such as AP2M1 or other μ subunits of clathrin AP complexes, anda cyclophilin inhibitor [i.e., NIM-811 (Novartis) and DEBIO-025(Debiopharm)] and/or an alpha-glucosidase inhibitor [i.e., Celgosivir(Migenix)] and/or one or more agents from one or more of the otherclasses of HCV therapeutic agents discussed herein is used to treat HCVinfection.

Other agents that can be used in combination with inhibiting agents ofthe present disclosure that prevent the binding of at least one viralprotein containing a YXXΦ or dileucine motif to host proteins such asAP2M1 or other μ subunits of clathrin AP complexes include (i) agentstargeting NS5A, including, but not limited to, A-831 (ArrowTherapeutics), AZD2836 (Astra Zeneca), and agents in development byXTL/Presidio or BMS (see PCT publications WO 2006/133326 and WO2008/021928, incorporated herein by reference); (ii) agents targetingTBC1D20 and/or NS5A's interaction with TBC1D20 (see PCT publication WO2007/018692 and U.S. patent application Ser. No. 11/844,993,incorporated herein by reference), (iii) agents targeting NS4B′s GTPaseactivity (see PCT publication WO 2005/032329 and US patent applicationpublication 2006/0199174, incorporated herein by reference); (iv) agentsinhibiting membrane association mediated by the HCV amphipathic helices,such as those found in NS5A, NS4B, and NS5B (see PCT publication WO2002/089731, supra), (v) agents targeting PIP2 or BAAPP domains in HCVproteins, such as those found in NS4B and NS5A (see U.S. provisionalpatent application 60/057,188, supra); (vi) agents targeting HCV entry,assembly, or release, including antibodies to co-receptors; (vii) agentstargeting HCV NS3 helicase; (viii) siRNAs, shRNAs, antisense RNAs, orother RNA-based molecules targeting sequences in HCV; (ix) agentstargeting microRNA122 or other microRNAs modulating HCV replication; (x)agents targeting PD-1, PD-L1, or PD-L2 interactions or pathways (see USpatent application publications 2008/0118511, 2007/0065427,2007/0122378, incorporated herein by reference); and (xi) agentstargeting HCV amphipathic helix function, such as AH2 inhibitors.

In another embodiment of the present disclosure, an inhibiting agentthat prevents the binding of at least one viral protein containing aYXXΦ or dileucine motif to host proteins such as AP2M1 or other μsubunits of clathrin AP complexes is used in combination with one ormore drugs capable of treating an HIV infection to treat a patient thatis co-infected with HIV and HCV. In another embodiment of the presentdisclosure, an inhibiting agent that prevents the binding of at leastone viral protein containing a YXXΦ or dileucine motif to host proteinssuch as AP2M1 or other μ subunits of clathrin AP complexes is used incombination with one or more drugs capable of treating an HBV infectionto treat a patient that is co-infected with HBV and HCV. In anembodiment, an inhibiting agent that prevents the binding of at leastone viral protein containing a YXXΦ or dileucine motif to host proteinssuch as AP2M1 or other μ subunits of clathrin AP complexes is used incombination with a PD-L1 inhibitor to treat a viral infection.

As mentioned above, embodiments of the present include theadministration of an inhibiting agent identified herein (or by using anembodiment of the screen of the invention) in conjunction with at leastone additional therapeutic agent to treat a viral infection. Suitableadditional therapeutic agents include, but are not limited to,ribavirin; a nucleoside analog (e.g., levovirin, viramidine, etc.); anNS3 inhibitor; an NS5 inhibitor; an interferon; and a side effectmanagement agent.

In an embodiment, the at least one additional suitable therapeutic agentincludes ribavirin. Ribavirin,1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide, available from ICNPharmaceuticals, Inc., Costa Mesa, Calif., is described in the MerckIndex, compound No. 8199, Eleventh Edition. Its manufacture andformulation is described in U.S. Pat. No. 4,211,771. The disclosure alsocontemplates use of derivatives of ribavirin (see, e.g., U.S. Pat. No.6,277,830).

In an embodiment, the at least one additional suitable therapeutic agentincludes levovirin. Levovirin is the L-enantiomer of ribavirin, andexhibits the property of enhancing a Th1 immune response over a Th2immune response. Levovirin is manufactured by ICN Pharmaceuticals.

In an embodiment, the at least one additional suitable therapeutic agentincludes viramidine. Viramidine is a 3-carboxamidine derivative ofribavirin, and acts as a prodrug of ribavirin. It is efficientlyconverted to ribavirin by adenosine deaminases.

Nucleoside analogs that are suitable for use in a combination therapyinclude, but are not limited to, ribavirin, levovirin, viramidine,isatoribine, an L-ribofuranosyl nucleoside as disclosed in U.S. Pat. No.5,559,101 and encompassed by Formula I of U.S. Pat. No. 5,559,101 (e.g.,1-β-L-ribofuranosyluracil, 1-β-L-ribofuranosyl-5-fluorouracil,1-β-L-ribofuranosylcytosine, 9-β-L-ribofuranosyladenine,9-β-L-ribofuranosylhypoxanthine, 9-β-L-ribofuranosylguanine,9-β-L-ribofuranosyl-6-thioguanine,2-amino-α-L-ribofuranl[1′,2′:4,5]oxazoline, O₂,O₂-anhydro-1-α-L-ribofuranosyluracil, 1-α-L-ribofuranosyluracil,1-(2,3,5-tri-O-benzoyl-α-ribofuranosyl)-4-thiouracil,1-α-L-ribofuranosylcytosine, 1-α-L-ribofuranosyl-4-thiouracil,1-α-L-ribofuranosyl-5-fluorouracil,2-amino-β-L-arabinofurano[1′,2′:4,5]oxazoline,O₂,O₂-anhydro-β-L-arabinofuranosyluracil, 2′-deoxy-β-L-uridine,3′5′-Di-O-benzoyl-2′deoxy-4-thio β-L-uridine, 2′-deoxy-β-L-cytidine,2′-deoxy-β-L-4-thiouridine, 2′-deoxy-β-L-thymidine,2′-deoxy-β-L-5-fluorouridine, 2′,3′-dideoxy-β-L-uridine,2′-deoxy-β-L-5-fluorouridine, and 2′-deoxy-β-L-inosine); a compound asdisclosed in U.S. Pat. No. 6,423,695 and encompassed by Formula I ofU.S. Pat. No. 6,423,695; a compound as disclosed in U.S. PatentPublication No. 2002/0058635, and encompassed by Formula 1 of U.S.Patent Publication No. 2002/0058635; a nucleoside analog as disclosed inWO 01/90121 A2 (Idenix); a nucleoside analog as disclosed in WO02/069903 A2 (Biocryst Pharmaceuticals Inc.); a nucleoside analog asdisclosed in WO 02/057287 A2 or WO 02/057425 A2 (both Merck/Isis); andthe like.

In an embodiment, the at least one additional suitable therapeutic agentcan include HCV NS3 inhibitors. Suitable HCV non-structural protein-3(NS3) inhibitors include, but are not limited to, a tri-peptide asdisclosed in U.S. Pat. Nos. 6,642,204; 6,534,523; 6,420,380; 6,410,531;6,329,417; 6,329,379; and 6,323,180 (Boehringer-Ingelheim); a compoundas disclosed in U.S. Pat. No. 6,143,715 (Boehringer-Ingelheim); amacrocyclic compound as disclosed in U.S. Pat. No. 6,608,027(Boehringer-Ingelheim); an NS3 inhibitor as disclosed in U.S. Pat. Nos.6,617,309; 6,608,067; and 6,265,380 (Vertex Pharmaceuticals); anazapeptide compound as disclosed in U.S. Pat. No. 6,624,290 (Schering);a compound as disclosed in U.S. Pat. No. 5,990,276 (Schering); acompound as disclosed in Pause et al. (2003); NS3 inhibitor BILN 2061(Boehringer-Ingelheim; Lamarre et al. (2002), and Lamarre et al. (2003);NS3 inhibitor VX-950 (Vertex Pharmaceuticals; Kwong et al. (2003); NS3inhibitor SCH6 (Abib et al. (2003); Program and Abstracts of the 54^(th)Annual Meeting of the American Association for the Study of LiverDiseases (AASLD, 2003); any of the NS3 protease inhibitors disclosed inWO 99/07733, WO 99/07734, WO 00/09558, WO 00/09543, WO 00/59929, or WO02/060926 (e.g., compounds 2, 3, 5, 6, 8, 10, 11, 18, 19, 29, 30, 31,32, 33, 37, 38, 55, 59, 71, 91, 103, 104, 105, 112, 113, 114, 115, 116,120, 122, 123, 124, 125, 126, and 127 disclosed in the table of pages224-226 in WO 02/060926); an NS3 protease inhibitor as disclosed in anyone of U.S. Patent Publication Nos. 2003/019067, 2003/0187018, and2003/0186895; and the like.

In an embodiment, the NS3 inhibitor used in a combination therapy of theinvention is a member of the class of specific NS3 inhibitors, e.g., NS3inhibitors that inhibit NS3 serine protease activity and that do notshow significant inhibitory activity against other serine proteases suchas human leukocyte elastase, porcine pancreatic elastase, or bovinepancreatic chymotrypsin, or cysteine proteases such as human livercathepsin B.

In an embodiment, the at least one additional suitable therapeutic agentincludes NS5B inhibitors. Suitable HCV non-structural protein-5 (N55;RNA-dependent RNA polymerase) inhibitors include, but are not limitedto, a compound as disclosed in U.S. Pat. No. 6,479,508(Boehringer-Ingelheim); a compound as disclosed in any of InternationalPatent Application Nos. PCT/CA02/01127, PCT/CA02/01128, andPCT/CA02/01129, all filed on Jul. 18, 2002 by Boehringer Ingelheim; acompound as disclosed in U.S. Pat. No. 6,440,985 (ViroPharma); acompound as disclosed in WO 01/47883, e.g., JTK-003 (Japan Tobacco); adinucleotide analog as disclosed in Zhong et al. (2003); abenzothiadiazine compound as disclosed in Dhanak et al. (2002); an NS5Binhibitor as disclosed in WO 02/100846 A1 or WO 02/100851 A2 (bothShire); an NS5B inhibitor as disclosed in WO 01/85172 A1 or WO 02/098424A1 (both Glaxo SmithKline); an NS5B inhibitor as disclosed in WO00/06529 or WO 02/06246 A1 (both Merck); an NS5B inhibitor as disclosedin WO 03/000254 (Japan Tobacco); an NS5B inhibitor as disclosed in EP 1256,628 A2 (Agouron); JTK-002 (Japan Tobacco); JTK-109 (Japan Tobacco);and the like.

In an embodiment, the NS5 inhibitor used in the combination therapies ofthe invention is a member of the class of specific NS5 inhibitors, e.g.,NS5 inhibitors that inhibit NS5 RNA-dependent RNA polymerase and thatlack significant inhibitory effects toward other RNA dependent RNApolymerases and toward DNA dependent RNA polymerases.

In an embodiment, the at least one additional therapeutic agent is aninterferon, e.g., interferon-alpha (IFN-α). Any known IFN-α. can be usedin the treatment methods of the invention. The term “interferon-alpha”as used herein refers to a family of related polypeptides that inhibitviral replication and cellular proliferation and modulate immuneresponse. The term “IFN-α” includes naturally occurring IFN-α; syntheticIFN-α; derivatized IFN-α (e.g., PEGylated IFN-α, glycosylated IFN-α, andthe like); and analogs of naturally occurring or synthetic IFN-α;essentially any IFN-α that has antiviral properties, as described fornaturally occurring IFN-α.

Suitable a interferons include, but are not limited to,naturally-occurring IFN-α (including, but not limited to, naturallyoccurring IFN-α2a, IFN-α2b); recombinant interferon α-2b such asIntron-A interferon available from Schering Corporation, Kenilworth,N.J.; recombinant interferon α-2a such as Roferon interferon availablefrom Hoffmann-La Roche, Nutley, N.J.; recombinant interferon α-2C suchas Berofor α2 interferon available from Boehringer IngelheimPharmaceutical, Inc., Ridgefield, Conn.; interferon α-n1, a purifiedblend of natural α interferons such as Sumiferon available fromSumitomo, Japan or as Wellferon interferon α-n1 (INS) available from theGlaxo-Wellcome Ltd., London, Great Britain; and interferon α-n3 amixture of natural α interferons made by Interferon Sciences andavailable from the Purdue Frederick Co., Norwalk, Conn., under theAlferon tradename.

The term “IFN-α” also encompasses consensus IFN-α. Consensus IFN-α (alsoreferred to as “CIFN” and “IFN-con” and “consensus interferon”)encompasses, but is not limited to, the amino acid sequences designatedIFN-con₁, IFN-con₂ and IFN-con₃ which are disclosed in U.S. Pat. Nos.4,695,623 and 4,897,471; and consensus interferon as defined bydetermination of a consensus sequence of naturally occurring interferonalphas (e.g., Infergen™, InterMune, Inc., Brisbane, Calif.). IFN-con₁ isthe consensus interferon agent in the Infergen™ alfacon-1 product. TheInfergen™ consensus interferon product is referred to herein by itsbrand name (Infergen™) or by its generic name (interferon alfacon-1).DNA sequences encoding IFN-con may be synthesized as described in theaforementioned patents or other standard methods. In an embodiment, theat least one additional therapeutic agent is CIFN.

In an embodiment, fusion polypeptides comprising an IFN-α and aheterologous polypeptide can also be used in the combination therapiesof the invention. Suitable IFN-α fusion polypeptides include, but arenot limited to, Albuferon-alpha™ [a fusion product of human albumin andIFN-α; Human Genome Sciences; see, e.g., Osborn et al. (2002)]. Alsosuitable for use in the present disclosure are gene-shuffled forms ofIFN-α. See, e.g., Masci et al. (2003). Other suitable interferonsinclude), Multiferon (Viragen), Medusa Interferon (Flamel Technology),Locteron (Octopus), and Omega Interferon (Intarcia/BoehringerIngelheim).

The term “IFN-α” also encompasses derivatives of IFN-α that arederivatized (e.g., are chemically modified relative to the naturallyoccurring peptide) to alter certain properties such as serum half-life.As such, the term “IFN-α” includes glycosylated IFN-α; IFN-α derivatizedwith polyethylene glycol (“PEGylated IFN-α”); and the like. PEGylatedIFN-α, and methods for making same, is discussed in, e.g., U.S. Pat.Nos. 5,382,657; 5,981,709; and 5,951,974. PEGylated IFN-α encompassesconjugates of PEG and any of the above-described IFN-α molecules,including, but not limited to, PEG conjugated to interferon alpha-2a(Roferon, Hoffman La-Roche, Nutley, N.J.), interferon alpha 2b (Intron,Schering-Plough, Madison, N.J.), interferon alpha-2c (Berofor Alpha,Boehringer Ingelheim, Ingelheim, Germany); and consensus interferon asdefined by determination of a consensus sequence of naturally occurringinterferon alphas (Infergen™, InterMune, Inc., Brisbane, Calif.).

In an embodiment, the IFN-α polypeptides can be modified with one ormore polyethylene glycol moieties, i.e., PEGylated. The PEG molecule ofa PEGylated IFN-α polypeptide is conjugated to one or more amino acidside chains of the IFN-α polypeptide. In an embodiment, the PEGylatedIFN-α contains a PEG moiety on only one amino acid. In anotherembodiment, the PEGylated IFN-α contains a PEG moiety on two or moreamino acids, e.g., the IFN-α contains a PEG moiety attached to two,three, four, five, six, seven, eight, nine, or ten different amino acidresidues. IFN-α may be coupled directly to PEG (i.e., without a linkinggroup) through an amino group, a sulfhydryl group, a hydroxyl group, ora carboxyl group.

To determine the optimum combination of an inhibiting agent, such asPKC-412, erlotinib, sunitinib, and the like, with other anti-HCV agents,HCV replication assays and/or animal studies can be performed in thepresence of various combinations of the various anti-HCV agents.Increased inhibition of replication in the presence of an additionalagent (above that observed with monotherapy) is evidence for thepotential benefit of the combination therapy.

For example, HCV replication assays employing a luciferasereporter-linked HCV genome in the presence of various combinations ofPKC-412, erlotinib, sunitinib, and the like. In such assays, luciferaseactivity is directly proportional to HCV RNA genome replication.

In an embodiment, side effect management agents can be used in thetreatment methods of the invention, and these include agents that areeffective in pain management; agents that ameliorate gastrointestinaldiscomfort; analgesics, anti-inflammatories, antipsychotics,antineurotics, anxiolytics, and hematopoietic agents. In addition,embodiments of the invention contemplate the use of any compound forpalliative care of patients suffering from pain or any other side effectin the course of treatment with a subject therapy. Exemplary palliativeagents include acetaminophen, ibuprofen, other NSAIDs, H2 blockers, andantacids. In an embodiment, the disclosure provides a method oftreatment with agents that inhibit GAK and agents that inhibit AAK1. Inan embodiment, such co-treatment provides synergistic effects, such asantiviral effects.

Hosts Suitable for Treatment

Hosts suitable for treatment with an embodiment of the inhibiting agentor an embodiment of the method include hosts who are infected with viralinfection from clathrin AP binding viruses, Flaviviridae, Lentiviridae,HCV, HIV, co-infections, such as HCV/HIV co-infections, Flaviviridaeother than HCV, clathrin AP binding viruses other than Flaviviridae, orclathrin AP binding viruses other than HCV. As used herein, the termFlaviviridae includes any member of the family Flaviviridae, including,but not limited to, Dengue virus, including Dengue virus 1, Dengue virus2, Dengue virus 3, Dengue virus 4 (see, e.g., GenBank Accession Nos.M23027, M19197, A34774, and M14931); Yellow Fever Virus; West NileVirus; Japanese Encephalitis Virus; St. Louis Encephalitis Virus; BovineViral Diarrhea Virus (BVDV); and Hepatitis C Virus (HCV); and anyserotype, strain, genotype, subtype, quasispecies, or isolate of any ofthe foregoing. Where the Flaviviridae is HCV, the HCV is any of a numberof genotypes, subtypes, or quasispecies, including, e.g., genotype 1,including 1a and 1b, 2, 3, 4, 6, etc. and subtypes (e.g., 2a, 2b, 3a,4a, 4c, etc.), and quasispecies.

Hosts suitable for treatment with embodiments of the present inventioninclude treatment failure patients. The term “treatment failurepatients” (or “treatment failures”) as used herein generally refers toHCV-infected patients who failed to respond to previous therapy for HCV(referred to as “non-responders”) or who initially responded to previoustherapy, but in whom the therapeutic response was not maintained(referred to as “relapsers”). The previous therapy generally can includetreatment with any antiviral agent other than an inhibiting agent of thepresent disclosure.

Hosts suitable for treatment with embodiments of the present disclosureinclude individuals who have been clinically diagnosed as infected withHCV. Individuals who are infected with HCV can be identified bydetecting HCV RNA in their blood, and/or having an anti-HCV antibody intheir serum.

Individuals who are clinically diagnosed as infected with HCV includenaive individuals (e.g., individuals not previously treated for HCV).

Hosts suitable for treatment with embodiments of the present disclosureinclude individuals who have any detectable HCV titer. The patient maybe infected with any HCV genotype (genotype 1, including 1a and 1b, 2,3, 4, 6, etc. and subtypes (e.g., 2a, 2b, 3a, etc.)), particularly adifficult to treat genotype such as HCV genotype 1 and particular HCVsubtypes and quasispecies.

Also suitable for treatment are HCV-positive hosts (as described above)who exhibit severe fibrosis or early cirrhosis (non-decompensated,Child's-Pugh class A or less), or more advanced cirrhosis(decompensated, Child's-Pugh class B or C) due to chronic HCV infectionand who are viremic despite prior antiviral treatment, or who have acontraindication to therapy with a known antiviral agent.

In an embodiment, HCV-positive hosts with stage 3 or 4 liver fibrosisaccording to the METAVIR scoring system, which is known in the art, aresuitable for treatment with the methods of the present disclosure. Inanother embodiment, hosts suitable for treatment with embodiments of thepresent disclosure are patients with decompensated cirrhosis withclinical manifestations, including patients with far-advanced livercirrhosis, including those awaiting liver transplantation. In stillanother embodiment, hosts suitable for treatment with embodiments of thepresent disclosure include patients with milder degrees of fibrosisincluding those with early fibrosis (stages 1 and 2 in the METAVIR,Ludwig, and Scheuer scoring systems; or stages 1, 2, or 3 in the Ishakscoring system, all of which are known in the art). In an embodiment,the method finds use in treatment of HCV post liver transplantation forHCV induced hepatocellular carcinoma. In an embodiment, this serves toprevent re-infection of the graft.

In an embodiment of the present disclosure, to help optimally selectpatients most likely to benefit from therapy, as well as to monitorefficacy of therapy—especially in the face of potential drug resistantmutant viruses—the use of appropriate diagnostic tests provided by thepresent invention can be of great benefit. For example, assessing thesensitivity of the specific virus found in a given patient to thecontemplated therapy can help identify the best match between candidatepatient and the corresponding appropriate therapy.

In an embodiment, the method provides treating patients infected withviruses as described herein, particularly those infected with HCV whoalso are afflicted with cancer, such as hepatic cancer.

Assays

The present disclosure also provides in vitro and cell-based methods ofscreening for antiviral agents. In one embodiment, the disclosureprovides methods for detecting interaction between the YXXΦ motif andhost proteins. In one embodiment, the present disclosure provides abinding assay to detect proteins or agents that inhibit binding of YXXΦor dileucine motifs to AP2M1 or other μ subunits of clathrin APcomplexes. For instance, the disclosure provides a method in whichcandidate agents as described herein are contacted with a Flaviviridaeand AP2M1 or other μ subunits of clathrin AP complexes. Binding of theFlaviviridae to the proteins is detected as is known in the art. Reducedactivity in the presence of a candidate agent relative to controlsindicates identification of a binding inhibitor. In another embodiment,cell-based binding assays are used. In another embodiment, cell assaysare used to screen for the effects on viral assembly or budding. In thisembodiment, candidate agents are contacted with a Flaviviridae and acell expressing AP2M1 or other μ subunits of clathrin AP complexes.Cellular effects, such as viral budding or assembly, or binding betweenthe Flaviviridae and cell can be assayed by methods known in the art. Inone embodiment, a replication assay is used. An example of a replicationassay is outlined in U.S. patent application publication 2011/0052536A1, which is expressly incorporated herein by reference. Reducedbinding, budding, or assembly and the like, of the virus in the presenceof the candidate agent relative to controls indicates identification ofa binding inhibitor or antiviral agent. In some embodiments, in vitromicrofluidics affinity assay, as known in the art, is also used toenable detection of weak and transient protein interactions.

In one embodiment, protein-fragment complementation assays, as describedherein and as are known in the art, are used to validate interactionbetween core and AP2M1. In another embodiment, a co-immunprecipitationassay is used to identify interaction between the proteins.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention and to set forth a clear understanding of theprinciples of the disclosure. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventors to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

Many variations and modifications may be made to the above-describedembodiments of the disclosure without departing substantially from thespirit and principles of the disclosure. All such modifications andvariations are intended to be included herein within the scope of thisdisclosure.

Example 1 Identification of a YXXΦ Motif within HCV Core

Inspection of the primary sequence of the HCV core protein reveals aconserved YIP(V/L) motif within the second domain (D2) of the protein(FIGS. 1A, D). This motif conforms to the YXXΦ sorting signal consensusrecognized by AP2M1 (Ohno, J Cell Sci 119:3719-3721, 2006; Nakatsu etal., Cell Struct Funct 28:419-429, 2003; Owen et al., Ann Rev Cell DevelBiol 20:153-191, 2004).

Core Binds AP2M1

Interactions of sorting signals with clathrin adaptors and endocyticcomponents are typically weak (Kd of binding at a μM range), transient(Nakatsu et al., Cell Struct Funct 28:419-429, 2003; Aguilar et al., JBiol Chem 276:13145-13152, 2001), and involve membrane proteins, thusdifficult to study by standard technologies (Cusick et al., Hum MolGenet 2005; Bailer Curr Opin Microbiol 12:453-459, 2009). To determinewhether HCV core binds AP2M1, proteomic platforms that overcome thesechallenges were used. In vitro microfluidics affinity assays are basedon mechanical trapping of molecular interactions (MITOMI), whicheliminates the off-rate problem facing current platforms, and thusallows studying weak and transient interactions, with nanoliter proteinconsumption (Maerkl et al., Science 315:233-237, 2007; Einav et al., NatBiotech 26:1019-1027, 2008; Gerber et al., Nat Meth 6:71-74, 2009). Amicrofluidics format that enables a high fidelity analysis ofprotein-protein interactions (P-PIs) was used (Gerber et al., Nat Meth6:71-74, 2009). In vitro protein expression in the presence ofmicrosomal membranes and binding experiments with MITOMI were performedessentially as described (Maerkl et al., Science 315:233-237, 2007;Einav et al., Nat Biotech 26:1019-1027, 2008; Gerber et al., Nat Meth6:71-74, 2009) (FIG. 9). These assays detected binding of AP2M1 to core.The degree of binding correlated with increasing core concentration(FIG. 1F). Background binding of AP2M1 to a control HCV protein, NS3,was 4-20 fold lower and did not increase significantly with increasedprotein concentration.

To determine whether the core-AP2M1 interaction occurs in cells,protein-fragment complementation assays (PCAs) based on reversiblereconstitution of a Gaussia princeps luciferase reporter was used (FIG.2A). The current format was optimized for improved signal, thusproviding a highly sensitive means for measuring challenging P-PIs(Cassonnet et al., Nat Meth 8:990-992, 2011). Significant luciferasesignal was detected in Huh-7.5 cells cotransfected with plasmidsencoding the two reporter fragments fused to the prey and bait proteins(GLuc1-AP2M1 and GLuc2-core). Background levels of binding were detectedin cells cotransfected with either the GLuc1-AP2M1 or GLuc2-coreconstructs and the empty reciprocal vector, the two empty GLuc vectors,or GLuc1 fused to three unrelated proteins (SPIRE, RAC1, and ARPC). Theapparent affinity of AP2M1 to core was higher than to transferrinreceptor (TFR), a host cargo protein harboring a YXXΦ signal, known tobe recognized by AP2M1 (Gminard et al., Traffic 5, 181-193, 2004) (FIG.2B). Binding was not genotype-specific, as core proteins derived fromeither the 2a (Lindenbach et al., Science 309:623-626, 2005) or 1b(Lohmann et al., Science 285:110-113, 1999) genotypes demonstratedcomparable levels of AP2M1 binding. Furthermore, there were nosignificant differences in core binding to the two isoforms ofAP2M1(a/b) (FIG. 2B). Comparable results were demonstrated in Huh-7.5(human hepatoma derived) cells, representing the most relevant cellmodel (FIG. 2B), and 293T cells (data not shown). Binding appearedspecific, as increasing concentrations of free core or AP2M1, but notnuclear export signal-interacting protein (NESI), a control proteininvolved in mediating hepatitis D virus assembly (Wang et al., J Virol79:8113-8120, 2005), progressively decreased core-AP2M1 binding (FIG.2C).

To determine whether core binds AP2M1 in the context of authentic HCVinfection, co-immunoprecipitation assays in membrane fractions preparedfrom Huh-7.5 cells infected with cell culture-grown HCV (J6/JFH) wereperformed (Lindenbach et al., Science 309:623-626, 2005). AP2M1 couldbring down core when anti-AP2M1 antibodies but not IgG controls wereadded to the membrane lysates. Binding was significantly augmented bycalyculin A [an inhibitor of AP2M1 dephosphorylation, which “locks”AP2M1 in its YXXΦ binding active conformation (Ricotta et al., J BiolChem 283:5510-5517, 2008)] (FIG. 2D). Binding in reciprocal conditionswas similarly demonstrated (FIG. 2D). Colocalization of core with AP2M1in Huh-7.5 cells 72 hr following electroporation with J6/JFH HCV RNA wasalso investigated. Quantitative confocal immunofluorescence (IF)analysis revealed extensive colocalization of core and AP2M1 in thesecells (with 67±6% colocalization of AP2M1 stained puncta with core)(FIG. 2E).

Core's YXXΦ Motif in AP2M1 Binding and HCV Assembly

Using a series of point mutations (FIG. 1D), whether AP2M1 binding ismediated by core's YXXΦ motif was tested. A Y136A core mutation reducedAP2M1 binding measured by PCAs by ˜10 fold compared with wild type (WT)core, whereas a V(Φ)139A mutation caused less inhibition of binding(FIG. 3A). To study the role of core's YXXΦ motif in HCV infection,these mutations were introduced into the J6/JFH(p7-Rluc2A) HCV genome—aRenilla luciferase-containing reporter virus that replicates andproduces high titers of virus in Huh-7.5 cells (Murray et al., J Virol81:10220-10231, 2007). Cells were electroporated with in vitrotranscribed RNA generated from each construct. HCV RNA replication ofthese viral mutants was comparable to that of the WT virus, as measuredby luciferase reporter gene-linked assays (FIG. 3B), and qRT-PCR (FIGS.11 and 12). In contrast, a polymerase-defective mutant,J6/JFH(p7-Rluc2A)-GNN, did not replicate. Luciferase assays in naivecells inoculated with supernatants derived from cells electroporatedwith viral genome harboring the Y136A core mutation measuredundetectable levels of extracellular infectivity. The V139A mutationdecreased infectivity by ˜1.5 logs compared to WT virus (FIG. 3C).Intracellular infectivity, measured in naive cells infected withclarified supernatants derived from lysed electroporated cells, mirroredthe diminished extracellular infectivity (FIG. 3D), suggesting thatcore's YXXΦ motif mediates virions assembly and not release. Essentiallyno infectious virus was produced either intra- or extracellularly byassembly (ΔE1-E2) or replication (GNN) defective controls. Infectivitytiters of WT virus measured by limiting dilution assays were comparableto those previously reported with this reporter system (Kopp et al., JVirol 84:1666-1673, 2010). Consistent with the luciferase assaysdescribed above, while the V139A core mutation decreased the extra- andintracellular infectivity titers by a ˜1-1.5 log compared with WT virus,an undetectable level of infectivity titers was measured with virusharboring the Y136A core mutation or E1-E2 deletion (FIG. 3E). Theeffect of core mutations on infectivity correlated with their effect onAP2M1 binding. To exclude the possibility that core's mutations affectedinfectivity by causing particle disassembly or production of defectivecore protein- and RNA-containing particles, production of noninfectiousparticles was determined. Detectable levels of HCV RNA and core proteinrelease were measured in supernatants of cells harboring replicatinggenomes by qRT-PCR and ELISA assays, respectively, as described (Murrayet al., J Virol 81:10220-10231, 2007; Kopp J Virol 84:1666-1673, 2010)(FIG. 3F, 3G). Nevertheless, the levels released by the Y136A and V139Acore mutants were not significantly higher than those released by theassembly-defective ΔE1-E2 mutant, suggesting that noninfectiousparticles were not produced. Core expression was not affected by themutations, as western analysis (FIG. 3H) and fluorescence microscopy(data is not shown) demonstrated protein expression at WT levels.Reversion of the infectivity phenotype was detected by luciferase assaysin Huh-7.5 cells infected with HCV harboring the Y136A and V139A coremutations following two weeks of passaging. This reversion coincidedwith the emergence of primary-site revertants by sequencing analysis.These results provide additional evidence for the requirement ofmaintaining a functional YXXΦ motif for supporting HCV replication.Together, these data suggest that the AP2M1 binding motif within core isrequired for viral assembly in vitro.

Core's YXXΦ Motif is Functionally Interchangeable with Other YXXΦSorting Signals

To determine whether core's YXXΦ motif is functionally interchangeablewith homologous signals, a V139L mutation, thus “swapping” the genotype2a core's sequence with that of genotype 1b was introduced. Similarly,the YTPL and YRRL sequences, known to mediate binding of HLA-DM (Ohno etal., J Bioll Chem 273:25915-25921, 1998) and thrombopoietin receptor(Hitchcock et al., Blood 112:2222-2231, 2008) to AP2M1, respectively,were used to substitute the core's YXXΦ sequence (FIG. 1D). Binding ofcore harboring these sequences to AP2M1 was either comparable to orgreater than that of WT core, as determined by PCAs (FIG. 3A). Thesemutations had no effect on HCV RNA replication (FIG. 3B). In correlationwith their biochemical phenotype, the intracellular and extracellularinfectivity of the V139L and YTPL core mutants were comparable to thatof WT virus (FIG. 3C, 3D). This functional interchangeability supportsthat core's YXXΦ motifs exerts its function via interactions with hostcell proteins. Despite its efficient binding to AP2M1 (FIG. 3A) andstability by western analysis (FIG. 3H), the YRRL mutant did not producedetectable levels of infectious virus (FIG. 3C, 3D). Interestingly, thismutant released HCV RNA and core protein into supernatants ofelectroporated cells at levels comparable to that of WT core, likelyreflecting production of noninfectious particles (FIG. 3F, 3G). The YRRLmutant may thus impact other function of core in infectious virusproduction that is independent of its binding to AP2M1.

AP2M1 in HCV Assembly

The functional relevance of AP2M1 to the HCV life cycle was determined.Stable Huh-7.5 clones harboring short hairpin RNA (shRNA) lentiviralconstructs targeting distinct regions in the AP2M1 gene or anon-targeting (NT) sequence were established. Effective suppression ofAP2M1 levels was achieved (FIG. 4A, 4B), without apparent cytostatic orcytotoxic effects. The effect of AP2M1 depletion on infectious virusproduction was studied in these clones following electroporation withJ6/JFH(p7-Rluc2A) RNA. AP2M1 knockdown had no effect on HCV RNAreplication as measured in these stable clones by luciferase assays(FIG. 4C) and qRT-PCR 72 hr following electroporation (data not shown).Supernatants collected at 72 hr postelectroporation were used toinoculate naive Huh-7.5 cells followed by luciferase assays at 72 hrpostinoculation. As shown in FIG. 4D, AP2M1 depletion reducedextracellular infectivity by >2 logs compared with NT control.Intracellular infectivity, measured in naive cells infected withclarified supernatants derived from lysed electroporated cells, mirroredthe diminished extracellular infectivity(FIG. 4E) and correlated withthe degree of AP2M1 depletion. Measurements of intra- or extracellularinfectivity titers by limiting dilution assays demonstrated consistentresults (FIG. 4F). AP2M1 depletion did not increase production ofnoninfectious particles, as suggested by the levels of HCV RNA and coreprotein release measured in supernatants of cells by qRT-PCR and ELISAassays, respectively (FIG. 4G, 4H). Similar effects on infectious virusproduction were demonstrated in Huh-7.5 cells transiently depleted forAP2M1 by siRNAs and either electroporated with the J6/JFH(p7-Rluc2A) RNAor infected with culture grown J6/JFH virus (titer: 1.2×10⁵ TCID₅₀/ml)(Lindenbach et al., Science 309:623-626, 2005). The effects of silencingendogenous AP2M1 on infectious virus production were rescued by ectopicexpression of shRNA-resistant WT AP2M1 harboring a wobble mutationwithin the site targeted by the shRNA, largely excluding the possibilityof off-target effects causing the observed phenotype (FIG. 4I). Thestable and transient RNAi approaches thus both suggest that AP2M1 isimportant for efficient HCV assembly.

Disruption of Core-AP2M1 Binding Abolishes Recruitment of AP2M1 to LD,Alters the Sub-Cellular Localization of Core, and Core Colocalizationwith E2

To test the hypothesis that the defect in HCV assembly resulting fromYXXΦ core mutations or AP2M1 silencing correlates with alterations inthe sub-cellular localization of AP2M1 and/or core, a quantitativeconfocal immunofluorescence (IF) analysis was performed. 10-15 randomlychosen cells were analyzed for each category for the degree oflocalization of core or AP2M1 to various intracellular compartmentsusing ImageJ (JACoP) software and Manders' Colocalization Coefficients(MCC). The latter were chosen, as they strictly measure co-occurrenceindependent of signal proportionality (Dunn et al., Am J Physiol-CellPhysiol 300:C723-C742, 2011). Endogenous AP2M1 minimally colocalizedwith the LD marker, Bodipy, in naive Huh-7.5 cells, with 8.2% of LDstaining positive for AP2M1 (FIG. 5A, 5E). In contrast, infection withJ6/JFH virus appeared to significantly increase the localization ofAP2M1 to LD, with 40±8% of LD being AP2M1 positive (FIG. 5B, 5E)(p-value=0.0006). Similarly, and as previously described (Kopp et al.,J. Virol. 84:1666-1673, 2010; Boulant et al., Journal of GeneralVirology 88:2204-2213, 2007; Coller et al., PLoS pathogens8:e1002466-e1002466, 2012), core was partially localized to LD (37±14%of core positive LD) (FIG. 5C, 5E). Furthermore, the partialcolocalization of core with AP2M1 occurred in part on LD (FIG. 5D, 5F).

To test whether core is involved in mediating the increased localizationof AP2M1 to LD measured in HCV infected cells, AP2M1-mCherry wasoverexpressed either alone or in combination with WT core or Y136A coremutant by transfecting Huh-7.5 cells. LD were labeled with HCS LipidTOX(Invitrogen). Similarly to naive cells, AP2M1 was minimally localized toLD in Huh-7.5 cells overexpressing AP2M1 alone (8±2% of LD positive forAP2M1) (FIG. 5G, 5J). In contrast, as in infected cells, whenco-expressed with WT core, AP2M1 appeared to significantly accumulate atLD, with 51.8±20% of LD being AP2M1 positive (p-value=4.8×10⁻⁵) (FIG.5H, 5J). No such increase in colocalization was demonstrated, however,when AP2M1 was co-expressed with core harboring the Y136A mutation (with10% AP2M1 positive LD) (p-value=0.001) (FIG. 5I, 5J), suggesting thatcore's YXXΦ motif may mediate recruitment of AP2M1 to LD.

The effect of disruption of the core-AP2M1 interaction on corelocalization to LD, ER, and TGN and its colocalization with the E2envelope protein in cells electroporated with J6/JFH HCV RNA was tested.As previously shown, core localized to all these intracellularcompartments (Kopp et al., J. Virol. 84, 1666-1673, 2010; Boulant etal., Journal of General Virology 88, 2204-2213, 2007) (with percentcolocalization ranging from 30 to 45%). Interestingly, localization ofcore harboring the Y136A mutation to LD was significantly greater thanthat of WT core (78±13% vs. 32±16%, respectively, p-value=2.7×10⁻⁶)(FIG. 5K). Similarly, the percent colocalization of core with the LDmarker was dramatically increased from 25±10% in NT cells to 87±6.6%following silencing of AP2M1 (p-value=1.55×10⁻⁸) (FIG. 5L). Whileneither the Y136A core mutation nor AP2M1 depletion had an apparenteffect on core colocalization with the ER marker, calreticulin (FIG.14), both were associated with a significant decrease in corecolocalization with the TGN marker, TGN46 (FIG. 5M, 5N) and the E2envelope protein (FIG. 5O, 5P).

Together, these results suggest that core's interaction with AP2M1facilitates recruitment of AP2M1 to LD and that the observed defect inHCV assembly following disruption of the core-AP2M1 interaction isassociated with accumulation of core on LD, decreased corecolocalization with E2, and impaired core trafficking to TGN.

AAK1 and GAK Regulate Core-AP2M1 Binding and are Involved in HCVAssembly

Phosphorylation of T156 within AP2M1 by the serine/threonine kinasesAAK1 and GAK (Ricotta et al., Journal of Cell Biology 156, 791-795,2002; Korolchuk et al., Traffic 3, 428-439, 2002; Zhang et al., Traffic6, 1103-1113, 2005) stimulates binding of AP2M1 to cargo proteintyrosine signals and is transient due to dephosphorylation by PP2A(Ricotta et al., Journal of Biological Chemistry 283, 5510-5517, 2008)(FIG. 6A). Indeed, calyculin A (a PP2A inhibitor) augmented the bindingof core to AP2M1 (FIG. 2D) and a T156A AP2M1 mutation impaired it (FIG.6B). To study the effect of overexpression of AP2M1 harboring the T156Amutation on infectious HCV production, Huh-7.5 cells were transfectedwith plasmids encoding either WT or AP2M1 T156A mutant. 48 hrposttransfection cells were electroporated with J6/JFH(p7-Rluc2A) HCVRNA and subjected to HCV RNA replication and infectivity assays, asdescribed above. Overexpression of the T156A AP2M1 mutant had no effecton cellular viability and was dispensable for HCV RNA replication, yetsignificantly reduced extra- and intracellular infectivity compared withWT AP2M1 (FIG. 6C-E), consistent with a dominant negative effect on HCVassembly.

To study the hypothesis that AAK1 and GAK are involved in regulating theinteraction of AP2M1 with core, binding experiments were performed inHuh-7.5 cells depleted for AAK1 or GAK by siRNAs (FIG. 6F, 6G).Depletion of either AAK1 or GAK significantly decreased core-AP2M1binding compared to NT, as measured by PCAs (FIG. 6H), with no apparentcytotoxic effect (data not shown). AAK1 and GAK depleted cells were thenelectroporated with J6/JFH(p7-Rluc2A) HCV RNA. While depletion of eitherAAK1 or GAK had no cytotoxic effect and was dispensable for HCV RNAreplication, it significantly reduced intracellular and extracellularinfectivity (FIG. 6I-K). The effect of AAK1 and GAK on HCV assembly didnot result from their direct binding to core (FIG. 6L). These resultsprovide evidence for the involvement of AAK1 and GAK in the regulationof core-AP2M1 binding and in mediating HCV assembly. Moreover, theyvalidate the importance of AP2M1 for efficient HCV assembly by adominant-interfering approach, thus supporting the RNAi data.

Pharmacological Inhibitors of AAK1 and GAK Disrupt Core-AP2M1 bindingand HCV assembly

Analysis of heat maps and affinity assays of kinase inhibitors (Karamanet al., Nat Biotech 26, 127-132, 2008) revealed compounds, such assunitinib and PKC-412, which bind AAK1, and erlotinib which binds GAK,with high affinities (nM range) (Karaman et al., Nat Biotech 26,127-132, 2008) (FIG. 7A, 7B). These compounds inhibited core-AP2M1binding in a dose-dependent manner, as determined by PCAs (FIG. 7C),with half maximal inhibitory concentrations (IC50s) of ˜0.04-0.2 μM.When used to treat Huh-7.5 cells electroporated with theJ6/JFH(p7-Rluc2A) HCV genome, these compounds had a dramaticdose-dependent effect on extra—(FIG. 7D) and intracellular infectivity(FIG. 7E) at 72 hr (with half maximal effective concentration (EC50s) of0.15-1.8 μM) (FIG. 7B). There was no effect on HCV RNA replication andno apparent cellular toxicity (FIG. 7F, 7G). Indeed, the inhibitoryeffect on core-AP2M1 binding and infectivity was associated withreductions in phospho-AP2M1 levels in the relevant cells by westernanalysis (FIG. 7H,). Last, these compounds significantly inhibited viralinfection in Huh-7.5 cells infected with tissue culture grown HCV(titer: 6.3×10⁵ TCID₅₀/ml ) (FIG. 7I, 7B,). These results providepharmacological validation for the involvement of AAK1 and GAK inregulating the core-AP2M1 interaction and for AP2M1's role in HCVassembly. Furthermore, they provide candidate compounds targetingassembly.

Example 2 AAK1 and GAK Depletion or Pharmacological Inhibition AbrogateHIV-1 Replication

There is a substantial body of evidence to support that interactions ofAP1M1 and AP2M1 with HIV Gag and Env (gp41) mediate several criticalsteps along the assembly/release pathway (Batonick et al., Virology342:190-200, 2005; Camus et al., Molec Biol Cell 18:3193-3203, 2007;Berlioz-Torrent et al., J Virol 73:1350-1361, 1999; Byland et al., MolecBiol Cell 18:414-425, 2007; Wyss et al., J Virol 75:2982-2992, 2001;Ohno et al., Virology 238:305-315, 1997; Boge et al., J Biol Chem273:15773-15778, 1998; Egan et al., J Virol 70:6547-6556, 1996; Rowellet al., J Immunol 155:473-488, 1995; Lodge et al., EMBO J 16:695-705,1997; Deschambeault et al., J Virol 73:5010-5017, 1999). Mostimportantly, a tyrosine motif within Env mediates cell-to-cell spread(Gminard et al., Traffic 5, 181-193, 2004; Lindenbach et al., Science309, 623-626, 2005), a mechanism thought to account for ongoing HIVreplication despite ART (Sigal et al., Nature 477, 95-98, 2011),possibly via AP1M1 which sorts to basolateral membranes. However, therole of AAK1 and GAK in HIV infection has not been studied, and thesemechanisms have not been targeted pharmacologically.

To test the hypothesis that AAK1 and GAK are important for HIVinfection, HeLa-derived TZM-b1 cells were used, which express CD4, CCR5and CXCR4, as well as β-galactosidase and firefly luciferase reportergenes responsive to HIV transcription (Wei et al., Antimicrobial agentsand chemotherapy 46:1896-1905, 2002). Cells were transfected with siRNAstargeting AAK1, GAK or a NT sequence followed by infection with theinfectious HIV-1 NL4-3 clone (Adachi et al., J Virol 59:284-291, 1986),as described (Zhou et al., Cell Host & Microbe 4:495-504, 2008).Luciferase activity was measured at 96 hr postinfection. AAK1 and GAKdepletion significantly inhibited HIV replication (FIG. 10). To test theeffect of pharmacological inhibitors of AAK1 and GAK against HIV, HIV-1infected HeLa-derived TZM-b1 cells were treated daily with serialdilutions of sunitinib, erlotinib or PKC-412 for 4 days followed byluciferase assays. A significant dose response antiviral effect wasobserved with no effect on viability (FIG. 10). EC₅₀ of erlotininb was1.4±0.3 (P value-0.0058), sunitinib-0.8±0.4 (P value-0.1), andPKC-412-8.5±4 (P value-0.1). Since these studies were conducted 96 hrpostinfection, they assessed all stages of infection from entry torelease and spread (Zhou et al., Cell Host & Microbe 4:495-504, 2008),and therefore suggest that AAK1 and GAK are important for overall HIVreplication.

Methods

Plasmids.

ORFs encoding AP2M1, GAK, AAK1, TFR, SPIRE, RAC1, ARPC, and NESI werepicked from the Human ORFeome library of cDNA clones (Rual et al.,Genome research 14, 2128-2135, 2004) (Open biosystems) and recombinedinto either pcDNA-Dest40 (for C-terminal V5-his tagging), pGLuc (forGaussia Princeps luciferase fragment (Gluc) tagging) (Cassonnet et al.,Nat Methods 8, 990-992, 2011), and/or pCherry (for mCherry fluorescenceprotein tagging) vectors using gateway technology (Invitrogen). ORFsencoding T7-tagged full length core and NS3 were amplified fromdescribed vectors (Blight et al., Science 290, 1972-1974, 2000) andligated into pcDNA3.1 (Invitrogen). pFL-J6/JFH(p7-Rluc2A) (Murray etal., J Virol 81, 10220-10231, 2007) was a gift from Dr. C. M. Rice(Tscherne et al., J Virol 80, 1734-1741, 2006). The YXXΦ core mutationsand AP2M1 mutations were introduced into these plasmids by site-directedmutagenesis (using the QuikChange kit (Stratagene)).

Antibodies and Compounds.

See Tables 1 and 2.

RNAi and Rescue of Gene Silencing.

40-100 nM individual or pooled siRNAs (FIG. 8) were transfected intocells using silMPORTER (Upstate, Millipore) 48 hr prior to HCV RNAelectroporation. Five individual MISSION Lentiviral TransductionParticles (Sigma) harboring short hairpin RNAs (shRNAs) targetingvarious sites in the AP2M1 RNA and a control shRNA were used totransduce Huh-7.5 cells according to the manufacturer's protocol. RNAireagents used in this study are summarized in Table 3. AP2M1 rescue wasperformed by transduction of Huh-7.5 cells stably depleted for AP2M1with lentiviruses expressing shRNA-resistant AP2M1 48 hr prior toelectroporation with HCV genome.

Microfluidics Affinity Assays.

Device fabrication and design were done essentially as described (Maerklet al., Science 315, 233-237, 2007). V5-his-tagged human proteins andT7-tagged viral proteins were expressed off the chip by mammalian invitro transcription/translation mixture (TNT) (Promega) (in the presenceof microsomal membranes). The device was subjected to surfacepatterning, resulting in a circular area coated with biotinylatedanti-his antibodies within each unit cell (Einav et al., NatureBiotechnology 26, 1019-1027, 2008; Gerber et al., Nature methods 6,71-74, 2009). Protein-protein binding experiments were performed asdescribed (Gerber et al., Nature methods 6, 71-74, 2009). Briefly, humanbait proteins were loaded into the device and bound to the surfaceanti-his antibodies. Viral and human proteins were incubated in the chipand labeled with anti-T7-Cy3 and anti-V5-FITC antibodies, respectively.Interactions were trapped mechanically by MITOMI (Maerkl et al., Science315, 233-237, 2007; Einav et al., Nature Biotechnology 26, 1019-1027,2008; Gerber et al., Nature methods 6, 71-74, 2009). After a brief washto remove untrapped unbound material, the trapped protein complexes weredetected by an array scanner (Tecan). The ratio of bound viral prey toexpressed human bait protein was calculated for each data point bymeasuring the ratio of median signal of Cy3 to median signal of FITC.Protein concentration in lysates were determined by quantitative westernanalysis against standard curves of T7-tagged proteins. Experiments wereconducted at least three times, each time with >20 replicates. See FIG.10 for a detailed protocol.

Cell Cultures.

Huh-7.5 cells and 293T cells were maintained in Dulbecco's modifiedminimal essential medium (Gibco) supplemented with 1% L-glutamine(Gibco), 1% penicillin, 1% streptomycin (Gibco), 1× nonessential aminoacids (Gibco), and 10% fetal bovine serum (Omega Scientific). Cell lineswere passaged three times a week after treatment with 0.05%trypsin-0.02% EDTA and seeding at a dilution of 1:4.

Protein-Fragment Complementation Assays.

Binding assays were performed essentially as described (Cassonnet etal., Nat Methods. 8, 990-992, 2011). Combinations of plasmids encodingprey (A) and bait (B) proteins, each fused to a fragment of the GaussiaPrinceps luciferase protein (Gluc1 and Gluc2) or control vectors werecotransfected into 293T or Huh-7.5 cells plated in 96-well plates intriplicates. At 24 hr posttransfection, cells were lysed and subjectedto standard luciferase reporter gene assays using Renilla luciferaseassays system (Promega). Results were expressed either as luminescenceor luminescence ratio. The latter represents the average luminescencesignal detected in cells transfected with Gluc1-A and Gluc2-B divided bythe average of luminescence measured in control wells transfected withGluc1-A and an empty Gluc2 vector with those transfected with Gluc2-Band an empty Gluc1 vector. Competition assays and studies designed todetermine inhibitory effect of compounds or siRNAs were performed asabove, except that binding was measured in the presence of excess freeproteins, the inhibitors, or siRNAs, respectively. Experiments wereconducted at least three times in triplicates.

Co-Immunoprecipitations.

Membranes were prepared from ˜20×10⁶ Huh-7.5 cells infected with HCV(J6/JFH) (Lindenbach, et al., Science 309, 623-626, 2005), as previouslydescribed (Einav et al., J Virol 78, 11288-11295, 2004). Briefly, cellswere collected by trypsinization, washed once with PBS and resuspendedin HME buffer (20 mM HEPES [pH 7.4], 1 mM EDTA, 2 mM MgCl2),supplemented with phenylmethylsulfonyl fluoride to a final concentrationof 1 mM and a protease inhibitors cocktail (Sigma). Cells were lysed bytwo cycles of freeze-thaw in dry ice-ethanol and then passaged through a27.5-gauge needle 10 times. Nuclei were removed by centrifugation at250×g for 10 min, and the postnuclear supernatant was subjected toultracentrifugation at 100,000×g for 30 min to obtain the membranepreparation. All steps were done at 4° C. Total membrane proteins werediluted in 1 ml TDB buffer (2.5% Triton X-100, 25 mM triethanolamine-Cl[pH 8.6], 20 mM NaCl, 5 mM EDTA, 0.2% NaN3) (Einav et al., J Virol 78,11288-11295, 2004) and incubated for 30 min at 37° C. with 100 nMcalyculin-A or DMSO. Due to the weak and transient nature of theinteractions, 25 mM dithiobis-succinimidyl-propionate (DSP) cross-linker(Pierce) was added to allow covalent binding of the already boundinteracting proteins. Samples were incubated for 2 hr on ice. 1 M Triswas added to stop the DSP activity. Lysates were then clarified by 10min centrifugation at 1000×g, followed by 30 min ultracentrifugation ofthe supernatants at 100,000×g. Membrane pellets were resuspended in 100ml HME buffer (20 mM NaHEpes (PH 7.4), 1 mM EDTA (Ph 8), 2 mM MgCl2),and TDB buffer was added for a final volume of 1 ml. Samples wereincubated overnight with either anti-AP2M1 antibodies, anti-coreantibodies or IgG controls, and protein G magnetic beads (Millipore).Immunoprecipitates were analyzed by western blotting. Experiments wereconducted twice in duplicates.

In Vitro Transcription of Viral RNA and Transfection.

HCV RNA was generated and delivered into Huh-7.5 cells, as previouslydescribed (Lindenbach et al., Science 309, 623-626, 2005; Murray et al.,J Virol 81, 10220-10231, 2007). Briefly, RNA was synthesized from XbaIlinearized J6/JFH(p7-Rluc2A) template using the T7 MEGAscript kitaccording to the manufacturer's protocol (Ambion). Reaction mixtureswere incubated for 3 hr at 37° C. and then subjected to DNase treatmentfor 15 min at 37° C. Viral RNA was purified using the RNeasy kit(Qiagen). RNA was quantified by absorbance at 260 nm, and its qualitywas verified by agarose gel electrophoresis. Subconfluent Huh-7.5 cellswere trypsinized and collected by centrifugation at 700 g for 5 min. Thecells were then washed three times in ice-cold RNase-free PBS(BioWhittaker) and resuspended at 1.5*10⁷ cells/ml in PBS. 5 μg of thein vitro transcribed wild type or J6/JFH(p7-Rluc2A) mutant RNA was mixedwith 400 μl of washed Huh-7.5 cells in a 2 mm-gap cuvette (BTX) andimmediately pulsed (0.82 kV, five 99 μs pulses) with a BTX-830electroporator. After a 15 min recovery at 25° C., cells were diluted in30 ml of prewarmed growth medium and plated into 96, 24, 6-well and P100tissue culture plates.

HCV RNA Replication by Luciferase Assays.

HCV RNA replication was measured at 6-9 hr, and 72 hrpostelectroporation, as described (Murray et al., J Virol 81,10220-10231, 2007). Electroporated cells plated in quadruplicates in96-well plates were washed twice with PBS and lysed with 30 μl ofRenilla lysis buffer (Promega). Following 15 min of shaking at RT,luciferase activity was quantified using a Renilla luciferase substrate(Promega) and a Tecan luminometer (Tecan) according to themanufacturers' protocols.

HIV Replication Assays.

HeLa-derived TZM-b1 cells, which express CD4, CCR5 and CXCR4, as well asβ-galactosidase and firefly luciferase reporter genes responsive to HIVtranscription (Wei et al., Antimicrobial agents and chemotherapy 46,1896-1905, 2002) were used in these assays. Cells were transfected withsiRNAs targeting AAK1, GAK or a NT sequence followed by infection withthe infectious HIV-1 NL4-3 clone (Adachi et al., J Virol 59, 284-291,1986), as described (Zhou et al., Cell Host & Microbe 4, 495-504, 2008).Luciferase activity was measured at 96 hr postinfection. To test theeffect of pharmacological inhibitors of AAK1 and GAK against HIV, HIV-1infected HeLa-derived TZM-b1 cells were treated daily with serialdilutions of sunitinib, erlotinib or PKC-412 for 4 days followed byluciferase assays.

Extracellular Infectivity.

Culture supernatants of Huh-7.5 cells electroporated withJ6/JFH(p7-Rluc2A) RNA and plated in P100 dishes were harvested at 72 hrpostelectroporation, clarified (with a 0.22-μm-pore size filter) andused to infect naïve Huh-7.5 cells for 72 hr in triplicates before lysisin Renilla lysis buffer (Promega). Luciferase activity in 20 μl of celllysates was quantified as described above. To determine the effect oferlotinib, sunitinib, and PKC-412 on infectious virus production,electroporated cells were grown in the presence of the inhibitors withdaily medium changes for 72 hr prior to collection of supernatants orcell lysates. Results represent log 10 RLU values per 10 cm tissueculture dish. Experiments were repeated three times, each time withquadruplicates.

Intracellular Infectivity Assays.

As described by Murray et al. (J Virol 81, 10220-10231, 2007), 72 hrpostelectroporation with J6/JFH(p7-Rluc2A) RNA cells were trypsinized,collected by centrifugation, resuspended in 500 μl medium, lysed byfreeze-thaw cycles, and pelleted twice at 3,650×g. Clarifiedsupernatants diluted in complete medium were used to inoculate naiveHuh-7.5 cells in triplicates, followed by lysis and luciferase assays at72 hr. Results represent log 10 RLU values per 10 cm tissue culturedish. Experiments were repeated three times, each time withquadruplicates.

Virus Titration.

Extracellular and Intracellular titers were determined by limitingdilution assays based on immunohistochemical staining for core. 50%tissue culture infectious dose (TCID₅₀) was calculated, as described(Lindenbach et al., Science 309, 623-626, 2005). Results are expressedas TCID₅₀/ml. Minimal titers measured with the ΔE1-E2 mutant were usedfor background subtraction.

Core ELISA.

The concentration of released core protein was measured in clarifiedcell culture supernatants harvested at 72 hr postelectroporation byELISA (Cell Biolabs) against standard curves of recombinant coreantigen, according to the manufacturer's instructions.

Viability Assays.

Following 24, 48, and 72 hrs of treatment with inhibitory compounds orsilencing with siRNAs, cells were incubated for 2-4 hrs at 37° C. in thepresence of 10% AlamarBlue reagent (TREK Diagnostic Systems), asdescribed (Einav et al., Nature Biotechnology 26, 1019-1027, 2008).Plates were then scanned and fluorescence was detected by usingFLEXstation II 384 (Molecular Devices, Inc.).

Infection Studies.

6×10³ Huh-7.5 cells seeded in 96-well plates were infected intriplicates with cell culture-grown HCV J6/JFH (titer: 1.2×10⁵TCID₅₀/ml). Extracellular and intracellular infectivity were measured byfocus formation assays (Lindenbach et al., Science 309, 623-626, 2005)in naive Huh-7.5 cells infected with supernatants or clarified celllysates derived from the infected cells at 72 hr postinfection.

To determine the antiviral effect of erlotinib, sunitinib, and PKC-412,6×10³ Huh-7.5 cells seeded in 96-well plates were infected intriplicates with cell culture-grown HCV (J6/JFH(p7-Rluc2A)) (titer:6.3×10⁵ TCID₅₀/ml) with an MOI (multiplicity of infection) of 0.1. Sixhours after infection and daily thereafter, cells were washed and mediumwas replaced with medium containing serial dilutions of the inhibitorycompounds. At 72 hr, samples were subjected to viability assays,followed by standard luciferase assays (Promega).

Analysis of Revertants.

Huh-7.5 cells electroporated with J6/JFH(p7-Rluc2A) harboring the Y136Aor V139A mutations were propagated. Culture supernatants were harvestedevery few days and used to inoculate naive Huh-7.5 cells fordetermination of extracellular infectivity by luciferase assays 72 hrfollowing inoculation. Total cellular RNA was extracted from clonesdemonstrating reversion of the infectivity phenotypone (at day 8postelectroporation for Y136A clones and day 14 for V139A mutant clones)using TRIzol reagent (Invitrogen). Reverse transcription reaction andPCR amplification were performed using Superscript One-Step reversetranscriptase PCR (RT-PCR) kit (Invitrogen). A ˜1-kb segment harboringthe core coding sequence was amplified. The PCR products were purifiedfrom agarose gels by Ultra Clean 15 DNA purification kit (MoBio) andsubjected to automatic sequencing on an ABI Prism 377 DNA sequencer(Sequetech). Presence of primary site reversion was confirmed by twoindependent sequences using forward and reverse oligos.

RNA Extraction and qRT-PCR.

Total RNA was isolated from cells or 1 ml cell culture supernatantsusing TRIzol (Invitrogen) or QiaAmp UltraSens kit (Qiagen),respectively. qRT-PCRs mixtures were assembled in triplicates using 0.5μg or 4 μl RNA and High-Capacity RNA-to-cDNA (Applied Biosystems).TaqMan reagents are listed in Table 4. Amplification and analysis wereperformed using StepOnePlus Real-Time-PCR system (Applied Biosystems).S18 was used as a control.

Western Blot.

Cell lysates were subjected to western analyses using primary antibodiesfollowed by HRP-conjugated secondary antibodies. Bands intensity wasquantified using NIH Image.

Quantitative Immunofluorescence Confocal Microscopy.

Huh-7.5 cells were electroporated with J6/JFH HCV RNA or infected withculture grown J6/JFH virus, seeded onto coverslips, and incubated for 72hr at 37° C. Cells were fixed with 4% paraformaldehyde in PBS, washedwith PBS, permeabilized with 0.1% Triton X-100 in PBS for 5 min, andblocked for 1 hr in PBS containing 1% BSA. Fixed cells were incubatedwith primary antibodies against either the core protein, AP2M1, TGN46,calreticulin or E2 at room temperature for 1 hr (see Table 1). Secondaryantibodies (goat anti-rabbit Alexa Fluor 488, goat anti-mouse AlexaFluor 594, chicken anti-goat IgG Alexa Fluor 647, and goat anti-humanAlexa Fluor 647) (Invitrogen) were incubated for 1 hr at roomtemperature. LD staining with Bodipy-488/503 (Invitrogen) was performedas described (Kopp et al., J Virol 84, 1666-1673, 2010). Cover slipswere then washed three times in PBS, and mounted with ProLong Goldantifade reagent (Invitrogen). Alternatively, Huh-7.5 cells wereco-transfected with plasmids expressing AP2M1-YFP and/or Core-mCherry.LD were stained with HCS LipidTOX (Invitrogen). All slides were analyzedusing Zeiss LSM 510 confocal microscope. Colocalization was quantifiedin 10-15 randomly chosen cells from each sample using ImageJ (JACoP)colocalization Software and Manders' Colocalization Coefficients (MCC).Threshold values were determined using auto threshold (plugin, ImageJ).Only pixels whose red and green intensity values were both above theirrespective thresholds were considered to be pixels with colocalizedprobes. MCCs were then calculated as the fractions of total fluorescencein the region of interest that occurs in these “colocal” pixels (with ahigher value representing more colocalization). M2 coefficient valuesrepresented as mean percent colocalization are shown.

Statistical Analysis.

IC50s and EC50s were measured by fitting data to a three parameterlogistic curve, as described (Einav et al., Nature Biotechnology 26,1019-1027, 2008). P-values were calculated using one-tailed unpairedStudent's t test.

Example 3 The Effect of Erlotinib, Sunitinib, and PKC-412 on AP2M1Phosphorylation

To determine the effect of the inhibitory compounds on AP2M1phosphorylation, Huh-7.5 cells harboring J6/JFH(p7-Rluc2A) RNA weretreated daily with various concentrations of the compounds or with DMSO.Since AP2M1 phosphorylation is transient (due to the activity of thephosphatase PP2A (Ricotta et al., J Biol Chem 283:5510-5517, 2008), toallow capturing of the phosphorylated AP2M1 state, lysates were preparedat 72 hr following a 30 min incubation of the cells with 100 nM of thePP2A inhibitor, calyculin A (Cal-A) or a DMSO control. Samples were thensubjected to SDS-PAGE and blotting with antibodies targeting either thephosphorylated AP2M1 form or actin. Indeed, significantly lower ratiosof phosphorylated-AP2M1 to actin were measured in lysates prepared fromcells treated with either sunitinib, erlotinib or PKC-412, compared withthe DMSO control (FIG. 6H). Phospho-AP2M1 to actin ratios wereprogressively decreased by increasing concentrations of sunitinib anderlotinib in a dose-dependent manner. These results suggest that AAK1 orGAK are potently inhibited by these compounds in Huh-7.5 cells harboringinfectious HCV.

The Effect of the Compounds on HCV RNA Replication, IntracellularInfectivity, Extracellular Infectivity, and HCV Replication.

To determine the effect of erlotinib, sunitinib, and PKC-412 oninfectious virus production, Huh-7.5 cells were electroporated withJ6/JFH(p7-Rluc2A) HCV genome (Murray et al., J Virol 81:10220-10231,2007), plated in 6-well plates, and treated daily with serial dilutionsof the compounds or DMSO. At 72 hr postelectroporation, cellularviability was measured by alamarBlue-based assays followed by luciferaseassays for determination of HCV RNA replication. Cell culturesupernatants and lysates were collected at 72 hr from parallel samplesand used to inoculate naive Huh-7.5 cells for determination ofextracellular and intracellular infectivity, respectively. Luciferaseassays were performed in these cells at 72 hr post inoculation.

To determine the effect of these compounds on HCV replication, 6×10³Huh-7.5 cells seeded in 96-well plates were infected in triplicates withcell culture-grown HCV (J6/JFH(p7-Rluc2A)) tittered at 6.3×10⁵ TCID₅₀/mlwith an MOI (multiplicity of infection) of 0.1. At 6 hrpostelectroporation, cells were washed and medium was replaced withserial dilutions of the inhibitory compounds. Cells were treated dailyfor 72 hr and then subjected to viability assays followed by standardluciferase assays.

Alternatively, 6×10³ Huh-7.5 cells seeded in 96-well plates wereinfected in triplicates with cell culture-grown HCV J6/JFH (titer:1.2×10⁵ TCID₅₀/ml with an MOI of 0.1, and subjected to focus formationassays, as described (Lindenbach et al., Science 309:623-626, 2005).Following 72 hr of daily treatment with the compounds, cells were fixedin 4% formaldehyde and permeabilized with saponin. HCV core protein wasdetected with primary anti-core monoclonal and secondary goat anti-mouseAlexa 594-conjugated antibodies. Foci were counted under an invertedmicroscope.

Experiments were repeated twice, each with 4 replicates. Signal wasnormalized to samples grown in the presence of the correspondingconcentration of DMSO. EC50s were measured by fitting data to a threeparameter logistic curve using the formula Y=a+(b−a)/(1+10{circumflexover ( )}(X−c)) (a, b and c represent minimum binding, maximum bindingand logEC50, respectively)(BioDataFit, Chang Bioscience, Inc).

TABLE 1 Antibodies used in this study Antibodies Source Biotinylatedanti-penta-his Qiagen FITC-conjugated anti-V5 InvitrogenPhycoerythrin-conjugated anti-T7 tag Abcam Rabbit Anti-AAK1 Abcam Rabbitanti-AP2M1 Santa Cruz Biotechnology Goat anti-AP2M1 Santa CruzBiotechnology Rabbit anti-phospho-AP2M1 (T156) Cell signaling Rabbitanti-TGN46 Abcam Rabbit anti-calreticulin Enzo Life Sciences Mouseanti-GAK MBL international corporation Mouse anti-core AustralBiologicals Human anti-E2 (CBH5) Dr. Steven K Foung Mouse anti-actinSigma HRP-conjugated anti-mouse IgG Cell signaling HRP-conjugatedanti-rabbit IgG Cell signaling

TABLE 2 Compounds used in this study Compound Source PKC-412 SigmaSunitinib malate Sigma Erlotinib LC Laboratories Calyculin A CellSignaling

TABLE 3 RNAi used in this study RNAi type Target Catalogue# SourceSequence ON-TARGETplus SMARTpools AP2M1 LQ-008170-00-0002 DharmaconAP2M1 J-004233-05 Dharmacon GUUAAGCGGUCCAACAUUU AP2M1 J-004233-06Dharmacon GCGAGAGGGUAUCAAGUAU AP2M1 J-004233-07 DharmaconAGUUUGAGCUUAUGAGGUA AP2M1 J-004233-08 Dharmacon GAACCGAAGCUGAACUACANon-targeting D-001810-10-05 Dharmacon Non Available siRNAs AAK1 Ref #4Ambion GGUGUGCAAGAGAGAAAUCtt GAK Ref #5 Dharmacon AACGAAGGAACAGCUGAUUCANon-targeting D-001210-01-05 Dharmacon Non Available MISSION shRNAsAP2M1 TRCN0000060238 Sigma CCGGGTGGTCATCAAGTCCAACTTTCTCGA (″238″)GAAAGTTGGACTTGATGACCACTTTTTG AP2M1 TRCN0000060239 SigmaCCGGCACCAGCTTCTTCCACGTTAACTCGA (″239″) GTTAACGTGGAAGAAGCTGGTGTTTTTGAP2M1 TRCNO000060241 Sigma CCGGGCTGGATGAGATTCTAGACTTCTCGA (″241″)GAAGTCTAGAATCTCATCCAGCTTTTTG AP2M1 TRCN0000060242 SigmaCCGGCATTTATGAAACTCGCTGCTACTCGAG (″242″) TAGCAGCGAGTTTCATAAATGTTTTTGNon-targeting SHC002V Sigma Non Available

TABLE 4 Taqman probes and primers used in this study Primer nameSequence or assay catalogue # HCV Forward CTTCACGCAGAAAGCGTCTAHCV Reverse CAAGCACCCTATCAGGCAGT HCV Taqman [6FAM]-TATGAGTGTCGTGCAGCCTC- MGB probe [MGB-NFQ] AP2M1 HS01037584_m1 GAKHS00178347_m1 AAK1 HS00208618_m1 S18 hs999999_m1All reagents listed in this table were purchased from AppliedBiosystems, Inc (ABI).

The invention claimed is:
 1. A method of inhibiting assembly or buddingof Dengue virus, comprising contacting a host cell that is infected withDengue virus with sunitinib or a pharmaceutically acceptable salt ofsunitinib, wherein the sunitinib inhibits adaptor-associated kinase 1(AAK1), thereby inhibiting assembly or budding of the Dengue virus. 2.The method of claim 1, wherein said sunitinib or pharmaceuticallyacceptable salt of sunitinib blocks binding of a viral structuralprotein to a host protein.
 3. The method of claim 1, wherein saidsunitinib or pharmaceutically acceptable salt of sunitinib competes witha viral structural protein for binding to a host protein.
 4. The methodof claim 1, wherein said sunitinib or pharmaceutically acceptable saltof sunitinib competes with a host protein for binding to a viralstructural protein.
 5. The method of claim 1, further comprisingcontacting said host cell with erlotinib, midostaurin, or apharmaceutically acceptable salt or combination thereof.
 6. The methodof claim 5, wherein the host cell is contacted with erlotinib or apharmaceutically acceptable salt of erlotinib.
 7. The method of claim 5,wherein the host cell is contacted with midostaurin or apharmaceutically acceptable salt of midostaurin.
 8. The method of claim1, wherein assembly or budding of said Flaviviridae is inhibited.